Transgenic plants expressing photorhabdus toxin

ABSTRACT

Novel polynucleotide sequences that encode insect toxins TcdA and TcbA have base compositions that differ substantially from the native genes, making them more similar to plant genes. The new sequences are suitable for use for high expression in both monocots and dicots. Transgenic plants with a genome comprising a nucleic acid of SEQ ID NO: 3 or SEQ ID NO:4 are insect resistant.

RELATED APPLICATION

[0001] This application claims priority from U.S. Provisional PatentApplication Ser. No. 60/148,356, filed Aug. 11, 1999.

BACKGROUND OF THE INVENTION

[0002] As reported in WO98/08932, protein toxins from the genusPhotorhabdus have been shown to have oral toxicity against insects. Thetoxin complex produced by Photorhabdus luminescens (W-14), for example,has been shown to contain ten to fourteen proteins, and it is known thatthese are produced by expression of genes from four distinct genomicregions: tca, tcb, tcc, and tcd. WO98/08932 discloses nucleotidesequences for the native toxin genes.

[0003] Of the separate toxins isolated from Photorhabdus luminescens(W-14), those designated Toxin A and Toxin B are especially potentagainst target insect species of interest, for example corn rootworm.Toxin A is comprised of two different subunits. The native gene tcdA(SEQ ID NO:1) encodes protoxin TcdA (see SEQ ID NO:1). As determined bymass spectrometry, TcdA is processed by one or more proteases to provideToxin A. More specifically, TcdA is an approximately 282.9 kDA protein(2516 aa) that is processed to provide TcdAii, an approximately 208.2kDA (1849 aa) protein encoded by nucleotides 265-5811 of SEQ ID NO:1,and TcdAiii, an approximately 63.5 kDA (579 aa) protein encoded bynucleotides 5812-7551 of SEQ ID NO:1.

[0004] Toxin B is similarly comprised of two different subunits. Thenative gene tcbA (SEQ ID NO:2) encodes protoxin TcbA (see SEQ ID NO:2).As determined by mass spectrometry, TcbA is processed by one or moreproteases to provide Toxin B. More specifically, TcbA is anapproximately 280.6 kDA (2504 aa) protein that is processed to provideTcbAii, an approximately 207.7 kDA (1844 aa) protein encoded bynucleotides 262-5793 of SEQ ID NO:2 and TcbAiii, an approximately 62.9kDA (573 aa) protein encoded by nucleotides 5794-7512 of SEQ ID NO:2.

[0005] The native tcdA and tcbA genes are not well suited for high levelexpression in plants. They encode multiple destabilization sequences,mRNA splice sites, polyA addition sites and other possibly detrimentalsequence motifs. In addition, the codon compositions are not like thoseof plant genes. WO98/08932 gives general guidance on how the toxin genescould be reengineered to more efficiently expressed in the cytoplasm ofplants, and describes how plants can be transformed to incorporate thePhotorhabdus toxin genes into their genomes.

SUMMARY OF THE INVENTION

[0006] In a preferred embodiment, the invention provides novelpolynucleotide sequences that encode TcdA and TcbA. The novel sequenceshave base compositions that differ substantially from the native genes,making them more similar to plant genes. The new sequences are suitablefor use for high expression in both monocots and dicots, and thisfeature is designated by referring to the sequences as the “hemicot”criteria, which is set forth in detail hereinafter. Other importantfeatures of the sequences are that potentially deleterious sequenceshave been eliminated, and unique restriction sites have been built in toenable adding or changing expression elements, organellar targetingsignals, engineered protease sites and the like, if desired.

[0007] In a particularly preferred embodiment, the invention providespolynucleotide sequences that satisfy hemicot criteria and that comprisea sequence encoding an endoplasmic reticulum signal or similar targetingsequence for a cellular organelle in combination with a sequenceencoding TcdA or TdbA.

[0008] More broadly, the invention provides engineered nucleic acidsencoding functional Photorhabdus toxins wherein the sequences satisfyhemicot criteria.

[0009] The invention also provides transgenic plants with genomescomprising a novel sequence of the invention that imparts functionalactivity against insects.

BRIEF DESCRIPTION OF SEQUENCES

[0010] SEQ ID NO:1 is the native tcdA DNA sequence together with thecorresponding encoded amino acid sequence for TcdA.

[0011] SEQ ID NO:2 is the native tcbA DNA sequence together with thecorresponding encoded amino acid sequence for TcbA.

[0012] SEQ ID NO:3 is an artificial sequence encoding TcdA that issuitable for expression in monocot and dicot plants.

[0013] SEQ ID NO:4 is an artificial sequence encoding TdbA that issuitable for expression in monocot and dicot plants.

[0014] SEQ ID NO:5 is an artificial hemicot sequence that encodes the 21amino acid ER signal peptide of 15 kDa zein from Black Mexican Sweetmaize.

[0015] SEQ ID NO:6 is an artificial hemicot sequence that encodes forthe full-length native TcdA protein (amino acids 22-2537) fused to themodified 15 kDa zein endoplasmic reticulum signal peptide (amino acids1-21).

DETAILED DESCRIPTION

[0016] The native Photorhabdus toxins are protein complexes that areproduced and secreted by growing bacteria cells of the genusPhotorhabdus. Of particular interest are the proteins produced by thespecies Photorhabdus luminescens. The protein complexes have a molecularsize of approximately 1,000 kDa and can be separated by SDS-PAGE gelanalysis into numerous component proteins. The toxins contain nohemolysin, lipase, type C phospholipase, or nuclease activities. Thetoxins exhibit significant toxicity upon ingestion by a number ofinsects.

[0017] A unique feature of Photorhabdus is its bioluminescence.Photorhabdus may be isolated from a variety of sources. One such sourceis nematodes, more particularly nematodes of the genus Heterorhabditis.Another such source is from human clinical samples from wounds, seeFarmer et al. 1989 J. Clin. Microbiol. 27 pp. 1594-1600. Thesesaprohytic strains are deposited in the American Type Culture Collection(Rockville, Md.) ATCC #s 43948, 43949, 43950, 43951, and 43952, and areincorporated herein by reference. It is possible that other sourcescould harbor Photorhabdus bacteria that produce insecticidal toxins.Such sources in the environment could be either terrestrial or aquaticbased.

[0018] The genus Photorhabdus is taxonomically defined as a member ofthe Family Enterobacteriaceae, although it has certain traits a typicalof this family. For example, strains of this genus are nitrate reductionnegative, yellow and red pigment producing and bioluminescent. Thislatter trait is otherwise unknown within the Enterobacteriaceae.Photorhabdus has only recently been described as a genus separate fromthe Xenorhabdus (Boemare et al., 1993 Int. J. Syst. Bacteriol. 43,249-255). This differentiation is based on DNA-DNA hybridizationstudies, phenotypic differences (e.g., presence (Photorhabdus) orabsence (Xenorhabdus) of catalase and bioluminescence) and the Family ofthe nematode host (Xenorhabdus; Steinernematidae, Photorhabdus;Heterorhabditidae). Comparative, cellular fatty-acid analyses (Janse etal. 1990, Lett. Appl. Microbiol 10, 131-135; Suzuki et al. 1990, J. Gen.Appl. Microbiol., 36, 393-401) support the separation of Photorhabdusfrom Xenorhabdus.

[0019] Currently, the bacterial genus Photorhabdus is comprised of asingle defined species, Photorhabdus luminescens (ATCC Type strain#29999, Poinar et al., 1977, Nematologica 23, 97-102). A variety ofrelated strains have been described in the literature (e.g., Akhurst etal. 1988 J. Gen. Microbiol., 134, 1835-1845; Boemare et al. 1993 Int. J.Syst. Bacteriol. 43 pp. 249-255; Putz et al. 1990, Appl. Environ.Microbiol., 56, 181-186).

[0020] The following toxin producing Photorhabdus strains have beendeposited: strain accession number date of deposit W-14 ATCC 55397 Mar.5, 1993 WX1 NRRL B-21710 Apr. 29, 1997 WX2 NRRL B-21711 Apr. 29, 1997WX3 NRRL B-21712 Apr. 29, 1997 WX4 NRRL B-21713 Apr. 29, 1997 WX5 NRRLB-21714 Apr. 29, 1997 WX6 NRRL B-21715 Apr. 29, 1997 WX7 NRRL B-21716Apr. 29, 1997 WX8 NRRL B-21717 Apr. 29, 1997 WX9 NRRL B-21718 Apr. 29,1997 WX10 NRRL B-21719 Apr. 29, 1997 WX11 NRRL B-21720 Apr. 29, 1997WX12 NRRL B-21721 Apr. 29, 1997 WX14 NRRL B-21722 Apr. 29, 1997 WX15NRRL B-21723 Apr. 29, 1997 H9 NRRL B-21727 Apr. 29, 1997 Hb NRRL B-21726Apr. 29, 1997 Hm NRRL B-21725 Apr. 29, 1997 HP88 NRRL B-21724 Apr. 29,1997 NC-1 NRRL B-21728 Apr. 29, 1997 W30 NRRL B-21729 Apr. 29, 1997 WIRNRRL B-21730 Apr. 29, 1997 B2 NRRL B-21731 Apr. 29, 1997 ATCC 43948 ATCC55878 Nov. 5, 1996 ATCC 43949 ATCC 55879 Nov. 5, 1996 ATCC 43950 ATCC55880 Nov. 5, 1996 ATCC 53951 ATCC 55881 Nov. 5, 1996 ATCC 43952 ATCC55882 Nov. 5, 1996 DEPI NRRL B-21707 Apr. 29, 1997 DEP2 NRRL B-21708Apr. 29, 1997 DEP3 NRRL B-21709 Apr. 29, 1997 P. zealandrica NRRLB-21683 Apr. 29, 1997 P. hepialus NRRL B-21684 Apr. 29, 1997 HB-Arg NRRLB-21685 Apr. 29, 1997 HB Oswego NRRL B-21686 Apr. 29, 1997 Hb LewistonNRRL B-21687 Apr. 29, 1997 K-122 NRRL B-21688 Apr. 29, 1997 HMGD NRRLB-21689 Apr. 29, 1997 Indicus NRRL B-21690 Apr. 29, 1997 GD NRRL B-21691Apr. 29, 1997 PWH-5 NRRL B-21692 Apr. 29, 1997 Megidis NRRL B-21693 Apr.29, 1997 HF-85 NRRL B-21694 Apr. 29, 1997 A. Cows NRRL B-21695 Apr. 29,1997 MP1 NRRL B-21696 Apr. 29, 1997 MP2 NRRL B-21697 Apr. 29, 1997 MP3NRRL B-21698 Apr. 29, 1997 MP4 NRRL B-21699 Apr. 29, 1997 MP5 NRRLB-21700 Apr. 29, 1997 GL98 NRRL B-21701 Apr. 29, 1997 G1101 NRRL B-21702Apr. 29, 1997 GL138 NRRL B-21703 Apr. 29, 1997 GL155 NRRL B-21704 Apr.29, 1997 GL217 NRRL B-21705 Apr. 29, 1997 GL257 NRRL B-21706 Apr. 29,1997

[0021] All strains were deposited in accordance with the terms of theBudapest Treaty. Strains having accession numbers prefaced by “ATTC”were deposited on the indicated date in the American Type CultureCollection, 12301 Parklawn Drive, Rockville, Md. 20852 USA. Strainsprefaced by “NRRL” were deposited on the indicated date in theAgricultural Research Service Patent Culture Collection (NRRL), NationalCenter for Agricultural Utilization Research, ARS-USDA, 1815 NorthUniversity St., Peoria Ill. 61604 USA.

[0022] The present invention provides hemicot nucleic acid sequencesencoding toxins from any Photorhabdus species or strain that produces atoxin having functional activity. Hemicot nucleic acid sequencesencoding proteins homologous to such toxins are also encompassed by theinvention.

[0023] Several terms that are used herein have a particular meaning andare defined as follows:

[0024] By “functional activity” it is meant herein that the proteintoxins) function as insect control agents in that the proteins areorally active, or have a toxic effect, or are able to disrupt or deterfeeding, which may or may not cause death of the insect. When an insectcomes into contact with an effective amount of toxin delivered viatransgenic plant expression, formulated protein compositions), sprayableprotein compositions), a bait matrix or other delivery system, theresults are typically death of the insect, or the insects do not feedupon the source which makes the toxins available to the insects.

[0025] By “homolog” it is meant an amino acid sequence that isidentified as possessing homology to a reference Photorhabdus toxinpolypeptide amino acid sequence.

[0026] By “homology” it is meant an amino acid sequence that has asimilarity index of at least 33% and/or an identity index of at least26% to a reference Photorhabdus toxin polypeptide amino acid sequence,as scored by the GAP algorithm using the BlOsum 62 protein scoringmatrix Wisconsin Package Version 9.0, Genetics Computer Group GCG),Madison, Wis.).

[0027] By “identity” is meant an amino acid sequence that contains anidentical residue at a given position, following alignment with areference Photrhabdus toxin polypeptide amino acid sequence by the GAPalgorithm.

[0028] By the use of the term “Photorhabdus toxin” it is meant anyprotein produced by a Photorhabdus microorganism strain which hasfunctional activity against insects, where the Photorhabdus toxin couldbe formulated as a sprayable composition, expressed by a transgenicplant, formulated as a bait matrix, delivered via baculovirus, ordelivered by any other applicable host or delivery system.

[0029] By the use of the term “toxic” or “toxicity” as used herein it ismeant that the toxins produced by Photorhabdus have “functionalactivity” as defined herein.

[0030] By “substantial sequence homology” is meant either: a DNAfragment having a nucleotide sequence sufficiently similar to anotherDNA fragment to produce a protein having similar biochemical properties;or a polypeptide having an amino acid sequence sufficiently similar toanother polypeptide to exhibit similar biochemical properties.

[0031] As with other bacterial toxins, the rate of mutation of thebacteria in a population causes many related toxins slightly differentin sequence to exist. Toxins of interest here are those which produceprotein complexes toxic to a variety of insects upon exposure, asdescribed herein. Preferably, the toxins are active against Lepidoptera,Coleoptera, Homopotera, Diptera, Hymenoptera, Dictyoptera and Acarina.The inventions herein are intended to capture the protein toxinshomologous to protein toxins produced by the strains herein and anyderivative strains thereof, as well as any protein toxins produced byPhotorhabdus. These homologous proteins may differ in sequence, but donot differ in function from those toxins described herein. Homologoustoxins are meant to include protein complexes of between 300 kDa to2,000 kDa and are comprised of at least two 2) subunits, where a subunitis a peptide which may or may not be the same as the other subunit.Various protein subunits have been identified and are taught in theExamples herein. Typically, the protein subunits are between about 18kDa to about 230 kDa; between about 160 kDa to about 230 kDa; 100 kDa to160 kDa; about 80 kDa to about 100 kDa; and about 50 kDa to about 80kDa.

[0032] As discussed above, some Photorhabdus strains can be isolatedfrom nematodes. Some nematodes, elongated cylindrical parasitic worms ofthe phylum Nematoda, have evolved an ability to exploit insect larvae asa favored growth environment. The insect larvae provide a source of foodfor growing nematodes and an environment in which to reproduce. Onedramatic effect that follows invasion of larvae by certain nematodes islarval death. Larval death results from the presence of, in certainnematodes, bacteria that produce an insecticidal toxin which arrestslarval growth and inhibits feeding activity.

[0033] Interestingly, it appears that each genus of insect parasiticnematode hosts a particular species of bacterium, uniquely adapted forsymbiotic growth with that nematode. In the interim since this researchwas initiated, the name of the bacterial genus Xenorhabdus wasreclassified into the Xenorhabdus and the Photorhabdus. Bacteria of thegenus Photorhabdus are characterized as being symbionts ofHeterorhabditus nematodes while Xenorhabdus species are symbionts of theSteinernema species. This change in nomenclature is reflected in thisspecification, but in no way should a change in nomenclature alter thescope of the inventions described herein.

[0034] The peptides and genes that are disclosed herein are namedaccording to the guidelines recently published in the Journal ofBacteriology “Instructions to Authors” p. i-xii January 1996), which isincorporated herein by reference.

[0035] Transformation methods useful in carrying out the invention arewell known, and are described, for example, in WO98/08932.

[0036] Hemicot tcdA and tcbA

[0037] SEQ ID NO: 3 is the nucleotide sequence for an engineered tcdAgene in accordance with the invention. SEQ ID NO: 4 is the nucleotidesequence for an engineered tcbA gene in accordance with the invention.

[0038] The following Tables 1 and 2 identify significant features of theengineered tcdA and tcbA genes. TABLE 1 tcdA Feature nucleotides of SEQID NO:3 NcoI   1-6 HindIII  48-53 KpnI  246-254 sequence encoding 267-5798 TcbAii NheI  333-338 BglII 1215-1220 ClaI 2604-2609 PstI4015-4020 AgeI 5088-5093 MunI 5598-5603 XbaI 5778-5783 sequence encoding5799-7517 TcbAiii AflII 5853-5858 SphI 6439-6444 SfuI 7392-7397 SacI7519-7524 XhoI 7522-7527 StuI 7528-7533 NotI 7533-7538

[0039] TABLE 2 tcbA Feature nucleotides of SEQ ID NO:5 NcoI   1-6HindIII  48-53 KpnI  246-251 sequence encoding  267-5798 TcbAii NheI 333-338 BglII 1215-1220 ClaI 2604-2609 PstI 4015-4020 AgeI 5088-5093MunI 5598-5603 XbaI 5778-5783 sequence 5799-7517 encoding TcbAiii AflII5853-5858 SphI 6439-6444 SfuI 7392-7397 SacI 7519-7524 SfuI 7392-7397SacI 7519-7524 XhoI 7522-7527 StuI 7528-7533 NotI 7535-7540

[0040] It should be noted that the proteins encoded by theplant-optimized tcdA (SEQ ID NO:3) and tcbA (SEQ ID NO:5) differ fromthe native proteins by the addition of an Ala residue at position #2.This modification was made to accommodate the NcoI site which spans theATG start codon.

[0041] The following Table 3 compares the codon composition of theengineered tcdA gene of SEQ ID NO:3 and engineered tcbA gene of SEQ IDNO:5 with the codon compositions of the native genes, the typical dicotgenes, and maize genes. TABLE 3 % in % in SEQ SEQ amino ID % in ID % in% in % in acid codon NO:3 tcdA NO:5 tcbA dicot maize Ala GCT 62 21 69 4142 24 GCC 26 32 27 17 27 34 GCA 11 25 4 22 25 18 GCG 0 21 0 21 6 24 ArgAGG 48 0 60 2 25 26 CGC 22 36 18 16 11 24 AGA 20 11 15 6 30 15 CGT 11 397 57 21 11 CGG 0 7 0 13 4 15 CGA 0 8 0 6 8 9 Asn AAC 100 32 100 33 55 68AAT 0 68 0 67 45 32 Asp GAC 67 22 70 25 42 63 GAT 33 78 30 75 58 37 CysTGC 100 30 100 19 56 68 TGT 0 70 0 81 44 32 End TGA 100 0 100 0 33 59TAG 0 0 0 0 19 21 TAA 0 100 0 100 48 20 Gln CAA 65 61 74 53 59 38 CAG 3539 26 47 41 62 Glu GAG 100 24 98 36 51 71 GAA 0 76 2 64 49 29 Gly GGT 6737 64 44 33 20 GGC 32 36 36 22 16 42 GGA 1 20 0 19 38 19 GGG 0 8 0 16 1220 His CAC 62 40 72 31 46 62 CAT 38 60 28 69 54 38 Ile ATC 73 34 65 2437 58 ATT 27 51 35 59 45 28 ATA 0 15 0 17 18 14 Leu CTC 54 11 59 7 28 26TTG 29 17 25 32 26 15 CTT 16 9 15 7 19 17 TTA 0 18 0 19 10 5 CTG 0 32 029 9 29 CTA 0 13 0 7 8 8 Lys AAG 99 79 99 75 61 78 AAA 1 21 1 25 39 22Met ATG 100 100 100 100 100 100 Phe TTC 100 42 100 41 55 71 TTT 0 58 059 45 29 Pro CCA 74 30 91 26 42 26 CCT 22 28 7 20 32 22 CCC 4 14 3 7 1724 CCG 0 27 0 47 9 28 Ser TCC 47 19 55 11 18 23 TCT 35 15 30 15 25 15AGC 18 22 15 18 18 23 AGT 0 20 0 31 14 9 TCG 0 7 0 8 6 14 TCA 0 17 0 1719 16 Thr ACC 60 41 64 31 30 37 ACT 28 25 32 34 35 20 ACA 12 21 4 18 2721 ACG 0 13 0 18 8 22 Trp TGG 100 100 100 100 100 100 Tyr TAC 100 24 10019 57 73 TAT 0 76 0 81 43 27 Val GTC 69 27 73 11 20 31 GTG 21 17 22 2729 39 GTT 10 34 3 48 39 21 GTA 0 22 2 14 12 8

EXAMPLE 1

[0042] Design Of Plant Codon-Biased Genes Encoding W-14 Peptides TcbAand TcdA

[0043] A. Gene Design

[0044] The coding strands of the native DNA sequences of thePhotorhabdus W-14 genes encoding peptides TcbA and TcdA were scanned forthe presence of deleterious sequences such as the Shaw/Kamen RNAdestabilizing motif ATTTA, intron splice recognition sites, and poly Aaddition motifs. This was done using the Macvector Sequence AnalysisSoftware (Oxford Molecular Biology Group, Symantec Corp.), using acustom Nucleic Acid Subsequence File. The native sequence was alsosearched for runs of 4 or more of the same base.

[0045] Motif searching of the native W-14 tcbA and tcdA genes revealedthe presence of many potentially deleterious sequences in the proteincoding strands, as summarized in Table 4. Not shown, but also present,were many runs of four or more single residues (e.g. the native tcbAgene has 81 runs of four A's). TABLE 4 Native Poly A Gene ATTTA 5′Splice 3′ Splice Addition* RNAP II term. tcbA 18 7 17 46 0 tcdA 18 7 1377 1

[0046] Analyses of eukaryotic genes and plant genes in particular haveshown that CG & TA doublets are underrepresented, while the genes areenriched in CT & TG doublets. The sequences of the hemicot biased geneshave accordingly been adjusted to encompass these base compositions andto have G+C compositions of about 53%, similar to-many plant genes. Whencompared to the native W-14 tcbA and tcdA genes, the plant-biased geneshave a much more uniform G+C distribution.

[0047] Nucleotide changes to remove potentially deleterious sequenceswere chosen to simultaneously adjust the codon composition of the codingregion to more closely reflect that of plant genes. A framework forthese changes was provided by the codon bias tables prepared for maizeand dicot genes shown in Table 3.

[0048] Comparison of codon compositions of the native W-14 genes tomaize and dicot genes revealed that the W-14 genes contain a verydifferent preference set of the degenerate codons for the 18 amino acidsfor which there is a choice (Table 3). For each of 8 amino acids (Phe,Tyr, Cys, Arg, Asn, Lys, Glu, and Gly) in both W-14 genes, the mostabundant codon is different from the preferred codons found in eithermaize or dicot genes. One might expect that translational difficultieswould be encountered in efforts to produce in plants proteins (such asTcbA and TcdA) having high relative amounts of these amino acids frommRNAs having large numbers of nonpreferred codons. There is a markeddifference in distribution of the codon compositions specifying theother 10 amino acids. For His, Gln, Ile, Val, and Asp, thedicot-preferred codons are found as the most abundant ones in both W-14genes. For Leu, Thr, Ser, and Ala, the maize preferred codons are themost abundant codon choices found in the tcdA gene. In contrast, thetcbA gene contains only the CCG (Pro) maize-preferred codon as thehighest abundance choice.

[0049] In making the codon choices, doublet contents were considered, sothat adjacent codons preferably did not form CG or TA doublets (whichare underrepresented in eukaryotic genes; 1, 4), while CT or TG doublets(which are enriched in eukaryotic genes ibid.) were created whenpossible.

[0050] Choices were also made to utilize a diversity of codons for Met,Trp, Asn, Asp, Cys, Glu, His, Ile, Lys, Phe, Thr, and Tyr.

[0051] The sequences were also designed to encode unique 6-bprecognition sites for restriction enzymes, spaced about every 1200 bp.Finally, an additional codon (GCT; Ala) was inserted at the secondposition to encode an Nco I recognition site encompassing the ATG (Met)start codon. Additional recognition sites were included after the stopcodon to facilitate subsequent cloning steps into expression vectors.These features are set forth above in Tables 1 and 2.

[0052] The new tcdA and tcbA genes of SEQ ID NO:3 and SEQ ID NO:4 share73.5%, and 72.6% identity, respectively, to their native W-14counterparts (Wisconsin Genetics Computer Group, GAP algorithm).

[0053] B. Gene Synthesis

[0054] The complete synthesis of the plant codon-biased tcbA and tcdAgenes was performed under contract by Operon Technologies, Inc. (OPTI,Alameda, Calif.). Basically, chemically synthesized oligonucleotides ofappropriate sequence were assembled into DNA pieces about 500 baseslong. These were joined together end-to-end (presumably by means ofappropriately placed restriction enzyme sites) into four larger piecesof roughly 2 kilobase pairs (kbp) each; therefore each comprised about ¼of the entire coding region of the particular gene. DNA sequence of thepieces was confirmed at this step. If mistakes in sequence were present,the appropriate oligonucleotides were re-synthesized, and the assemblyprocess was repeated. Once gene fractional parts were sequence verified,they were assembled in pairs to make the gene halves, and again sequenceverified. Finally, the two halves were joined, and the sequences of thejunctions between the halves was verified. Therefore, each part of thenew gene was sequence verified at least twice.

[0055] It should be noted that attempts to express the native tcbA ortcdA genes in standard Escherichia coli cloning strains suggests thatproduction of these proteins is lethal. Lethality problems may beencountered if standard cloning vectors having leaky expression frominherent lacZ promoters are used to assemble these genes.

[0056] C. Addition of Endoplasmic Reticulum Targeting Peptide to TcdACoding Region

[0057] It is known to those in the field of plant gene expression thatproteins are specifically directed into the endoplasmic reticulum (ER)by means of a short signal peptide which is removed during or after thetransport process through the ER membrane. The mature (processed)protein is incorporated into the ER endomembrane or is released into theER lumen where the transported protein may be uniquely folded (aided bychaperonins), modified by glycosylation, accumulated in the vacuole, oradditionally translocated (by secretion). These processes are reviewedby Gomord and Faye [V. Gomord and L. Faye, (1996) Signals and mechanismsinvolved in intracellular transport of secreted proteins in plants.Plant Physiol. Biochem. 34:165-181] and by Bar-Peled et al. [M.Bar-Peled, D. C. Bassham, and N. V. Raikhel, (1996) Transport ofproteins in eukaryotic cells: more questions ahead. Plant Molec. Biology32:223-249]. It is also known that the subcellular recognitionmechanisms for an ER signal peptide are evolutionarily somewhatconserved, since the ER signal for a protein normally produced inmonocot (maize) cells is recognized and processed normally by dicot(tobacco) cells. This is exemplified by the maize 15 kDa zein ER signalpeptide [L. M. Hoffman, D. D. Donaldson, R. Bookland, K. Rashka, and E.M. Herman, (1987) Synthesis and protein body deposition of maize 15-kdzein in transgenic tobacco seeds. EMBO J. 6:3213-3221, and U.S. Pat. No.5,589,616]. Further, it is known that the ER signal peptide derived fromone protein can direct the translocation of a different protein if it isappropriately attached to the second protein by genetic engineeringmethods [D. C. Hunt and M. J. Chrispeels, (1991) The signal peptide of avacuolar protein is necessary and sufficient for the efficient secretionof a cytosolic protein. Plant Physiol. 96:18-25, and Denecke, J., J.Botterman, and R. Deblaere (1990) Protein secretion in plants can occurvia a default pathway. Plant Cell 2:51-59]. Therefore, one may expose aprotein in vivo to different biochemical environments by directing itsaccumulation in the cytosol (by not providing a signal peptidesequence), or in the ER/vacuole (by provision of an appropriate signalpeptide.)

[0058] The ER signal peptide of maize 15 kDa zein proteins is known tocomprise the first 20 amino acids encoded by the zein coding region. Twoexamples of such signal peptides the ER signal peptide of 15 kDa zeinfrom A5707 maize, NCBI Accession # M72708, and the ER signal peptide of15 kDa zein from Black Mexican Sweet maize, NCBI Accession #M13507.There is only a single amino acid difference (Ser vs Cys at residue 17)between these signal peptides.

[0059] SEQ ID NO:5 is a modified sequence coding the ER signal peptideof 15 kDa zein from Black Mexican Sweet maize. The modificationsembodied in this sequence were made to accommodate the differentmonocot/dicot codon usages and other sequence motif considerationsdiscussed above in the design of the plant-optimized tcdA coding region.The sequence includes an additional Ala residue at position #2 toaccommodate the NcoI site which spans the ATG start codon.

[0060] SEQ ID NO:6 gives a sequence coding for the full-length nativeTcdA protein (amino acids 22-2537) fused to the modified 15 kDa zeinendoplasmic reticulum signal peptide (amino acids 1-21).

EXAMPLE 2 Transformation of Tobacco with Agrobacterium Carrying PlasmidpDAB2041 Encoding Photorhabdus Toxins

[0061] A. Plasmid pDAB2041

[0062] Preparation of tobacco transformation vectors was accomplished inthree steps. First, a modified plant-optimized tcdA coding region wasligated into a tobacco plant expression cassette plasmid. In this step,the coding region was placed under the transcriptional control of apromoter functional in tobacco plant cells. RNA transcriptiontermination and polyadenylation were mediated by a downstream copy ofthe terminator region from the Agrobacterium nopaline synthase gene. Twoplasmids designed to function in this role are pDAB1507 and pDAB2006. Inthe second step, the complete gene comprised of the promoter, codingregion, and terminator region was ligated between the T-DNA borders ofan Agrobacterium binary vector, pDAB1542. Also positioned between theT-DNA borders was a plant selectable marker gene to allow selection oftransformed tobacco plant cells. In the third step, the engineeredbinary vector plasmid was conjugated from its E. coli host strain into adisabled Agrobacterium tumefaciens strain capable of transformingtobacco plant cells that regenerate into fertile transgenic plants.

[0063] It is a feature of plasmid pDAB1507 that any coding region havingan NcoI site at its 5′ end and a SacI site 3′ to the coding region, whencloned into the unique NcoI and SacI sites of pDAB1507, is placed underthe transcriptional control of an enhanced version of the CaMV 35Spromoter. It is also a feature of pDAB1507 that the 5′ untranslatedleader (UTR) sequence preceding the NcoI site comprises a modifiedversion of the 5′ UTR of the MSV coat protein gene, into which has beencloned an internally deleted version of the maize Adh1S intron 1.Additionally it is a feature of pDAB1507 that transcription terminationand polyadenylation of the mRNA containing the introduced coding regionare mediated by termination/Poly A addition sequences derived from thenopaline synthase (Nos) gene. Finally, it is a feature of pDAB1507 thatthe entire assembly of promoter/coding region/3′UTR can be obtained as asingle DNA fragment by cleavage at the flanking NotI sites.

[0064] It is a feature of plasmid pDAB2006 that any coding region havingan NcoI site at its 5′ end and a SacI site 3′ to the coding region, whencloned into the unique NcoI and SacI sites of pDAB2006, is placed underthe transcriptional control of the CaMV 35S promoter. It is also afeature of pDAB2006 that the 5′ untranslated leader (UTR) sequencepreceding the NcoI site comprises a polylinker. Additionally it is afeature of pDAB2006 that transcription termination and polyadenylationof the mRNA containing the introduced coding region are mediated bytermination/Poly A addition sequences derived from the nopaline synthase(Nos) gene. Finally, it is a feature of pDAB2006 that the entireassembly of promoter/coding region/3′UTR can be obtained as a single DNAfragment by cleavage at the flanking NotI sites.

[0065] It is a feature of pDAB1542 that any DNA fragment flanked by NotIsites can be cloned into the unique NotI site of pDAB1542, thus placingthe introduced fragment between the T-DNA borders, and adjacent to theneomycin phosphotransferase II (kanamycin resistance) gene.

[0066] To prepare a plant-expressible gene to produce the non-targetedTcdA protein in tobacco plant cells, DNA of a plasmid (pA0H_(—)4-OPTI)containing the plant-optimized tcdA coding region, (SEQ ID No:3) wascleaved with restriction enzymes NcoI and SacI, and the large 7550 bpfragment was ligated to similarly-cut DNA of plasmid pDAB1507 to produceplasmid pDAB2040. DNA of pDAB2040 was then digested with NotI, and the8884 bp fragment was ligated to NotI digested DNA of pDAB1542 to produceplasmid pDAB2041. This plasmid was then conjugated by triparental mating[Firoozabady, E., D. L. DeBoer, D. J. Merlo, E. L. Halk, L. N. Amerson,K. E. Rashka, and E. E. Murray (1987) Transformation of cotton(Gossypium hirsutum L.) by Agrobacterium tumefaciens and regeneration oftransgenic plants. Plant Molec. Biol. 10:105-116] from the hostEscherichia coli strain (XL1-Blue, Stratagene, La Jolla, Calif.), intothe nontumorigenic Agrobacterium tumefaciens strain EHA101S, which is aspontaneous streptomycin-resistant mutant of strain EHA101 (Hood, E. E.,G. L. Helmer, R. T. Fraley, and M. -D. Chilton (1986) The hypervirulenceof Agrobacterium tumefaciens A281 is encoded in a region of pTiBo542outside of T-DNA. J. Bacteriol. 168:1291-1301). Strain EHA101S(pDAB2041)was then used to produce transgenic tobacco plants that expressed theTcdA protein.

[0067] B. Plasmid pRK2013

[0068] To prepare a plant-expressible gene to produce the endoplasmicreticulum-targeted TcdA protein in tobacco plant cells, DNA of a plasmid(pA0H_(—)4-ER) containing the plant-optimized, ER-targeted tcdA codingregion, (SEQ ID No:6) was cleaved with restriction enzymes NcoI andSacI, and the large 7610 bp fragment was ligated to similarly-cut DNA ofplasmid pDAB2006 to produce plasmid pDAB1833. DNA of pDAB1833 was thendigested with NotI, and the 8822 bp fragment was ligated to NotIdigested DNA of pDAB1542 to produce plasmid pDAB2052. This plasmid wasthen conjugated by triparental mating from the host Escherichia colistrain (XL1-Blue), into the nontumorigenic Agrobacterium tumefaciensstrain EHA101S. Strain EHA101S(pDAB2052) was then used to producetransgenic tobacco plants that expressed the TcdA protein containing anamino terminus endoplasmic reticulum targeting peptide.

[0069] C. Transfer of Plasmid pDAB2041 Into Agrobacterium Strain EHA101S

[0070] Cultures of E. coli carrying the engineered Ti plasmid pDAB2041(plasmid containing the rebuilt Toxin A gene, tcdA), E. coli carryingthe plasmid pRK2013, and Agrobacterium strain EHA101S were grownovernight, then mixed 1:1:1 on plain LB medium solidified with agar andcultured in the dark at 28° C. Two days later, the lawn of bacteria wasscraped up with a loop, suspended in plain LB medium, vortexed, and thendiluted 1:10⁴, 1:10⁵, and 1:10⁶ fold in plain LB liquid medium. Aliquotsof these dilutions were spread on selective plates containing medium YEPplus erythromycin (100 mg/L) and streptomycin (250 mg/L) and grown at28° C. Two days later, single colonies were picked and streaked onto thesame medium, then spread to give single colonies. Single colonies werepicked again and streaked, then spread for single colonies. Singlecolonies were picked a third time, grown as streaks, then subjected to aquality analysis involving growth on lactose medium and chromogenicassay with Benedict's reagent. Of ten strains developed in this way, thefastest coloring colony was chosen for further work.

[0071] D. Transformation of Tobacco with Agrobacterium Carrying PlasmidpDAB2041

[0072] Tobacco transformation with Agrobacterium tumefaciens was carriedout by a method similar, but not identical, to published methods (RHorsch et al, 1988. Plant Molecular Biology Manual, S. Gelvin et al,eds., Kluwer Academic Publishers, Boston). To provide source tissue forthe transformation, tobacco seed (Nicotiana tabacum cv. Kentucky 160)were surface sterilized and planted on-the surface of TOB−, which is ahormone-free Murashige and Skoog medium (T. Murashige and F. Skoog,1962). A revised medium for rapid growth and bioassays with tobaccotissue culture. Plant Physiol. 75: 473-497) solidified with agar. Plantswere grown for 6-8 weeks in a lighted incubator room at 28-30° C. andleaves were collected sterilely for use in the transformation protocol.Approximately one cm² pieces were sterilely cut from these leaves,excluding the midrib. Cultures of the Agrobacterium strains (EHA101Scontaining pDAB2041), which had been grown overnight on a rotor at 28°C., were pelleted in a centrifuge and resuspended in sterile Murashige &Skoog salts, adjusted to a final optical density of 0.7 at 600 nm. Leafpieces were dipped in this bacterial suspension for approximately 30seconds, then blotted dry on sterile paper towels and placed right sideup on medium TOB+ (Murashige and Skoog medium containing 1 mg/L indoleacetic acid and 2.5 mg/L benzyladenine) and incubated in the dark at 28°C. Two days later the leaf pieces were moved to medium TOB+ containing250 mg/L cefotaxime (Agri-Bio, North Miami, Fla.) and 100 mg/L kanamycinsulfate (AgriBio) and incubated at 28-30° C. in the light. Leaf pieceswere moved to fresh TOB+ with cefotaxime and kanamycin twice per weekfor the first two weeks and once per week thereafter. Leaf pieces whichshowed regrowth of the Agrobacterium strain were moved to medium TOB+with cefotaxime and kanamycin, plus 100 mg/l carbenicillin (Sigma). Fourto six weeks after the leaf pieces were treated with the bacteria, smallplants arising from transformed foci were removed from this tissuepreparation and planted into medium TOB− containing 250 mg/L cefotaximeand 100 mg/L kanamycin in Magenta GA7 boxes (Magenta Corp., Chicago).These plantlets were grown in a lighted incubator room. After 3-4 weeksthe primary transgenic plants had rooted and grown to a size sufficientthat leaf samples could be analyzed for expression of protein from thetransgene. Twenty-five independent transgenic events were recovered assingle plants from the pDAB2041 transformation.

[0073] Eight independent lines expressing various levels of transgenicprotein from the T-DNA of pDAB2041 were propagated in vitro from leafpieces as follows. Twelve to sixteen approximately one cm² pieces weresterilely cut from leaves of each primary transgenic plant, excludingthe midrib and all naturally occurring edges. These leaf pieces wereplaced on medium TOB+ containing 250 mg/L cefotaxime and 100 mg/Lkanamycin, and cultured in the lighted incubator at 28-30° C. for 3-4weeks, at which time small plants could be cut from the proliferatingtissue mass. Several small plantlets from each transgenic line weremoved into Magenta boxes containing medium TOB− plus cefotaxime andkanamycin and allowed to root and grow. The proliferating tissue masswas further cultured on medium TOB+ with cefotaxime and kanamycin, andadditional plants could be cut out and grown up as needed.

[0074] Plants were moved into the greenhouse by washing the agar fromthe roots, transplanting into soil in 5½″ square pots, placing the potinto a Ziploc bag (DowBrands), placing plain water into the bottom ofthe bag, and placing in indirect light in a 30° C. greenhouse for oneweek. After one week the bag could be opened; the plants were fertilizedand allowed to grow further, until the plants were acclimated and thebag was removed. Plants were grown under ordinary warm greenhouseconditions (30° C., 16H light). Plants were suitable for sampling fourweeks post transplant.

EXAMPLE 3 Chacterization of Transgenic Tobacco Plants ExpressingPhotorhabdus Toxin that Confer Insect Control

[0075] A. Polyclonal Antibody Production

[0076] The E. coli produced recombinant TcdA protein was purified by aseries of column purification. The protein was sent to Berkley AntibodyCompany (Richmond, Calif.) for the production of antiserum in a rabbit.Inoculations with the antigen were initiated with 0.5 mg of proteinfollowed by four boosting injections of 0.25 mg each at about three weekintervals. The rabbit serum was tested by the standard Western analysisusing the recombinant TcdA protein as the antigen and enhancedchemi-luminescens, ECL method (Amersham, Arlington Heights, Ill.) Theantibodies (PAb-EA₀) were purified using a PURE I antibody purificationkit (Sigma, St. Luis, Mo.). PAb-EA₀ antibodies recognize the full-lengthTcdA and its processed components.

[0077] B. Expression of TcdA Protein in Tobacco

[0078] Protein was extracted from the leaf tissue of transformed andnon-transformed tobacco plants following the procedure describedimmediately below.

[0079] Two leaf disks of 1.4 cm in diameter were harvested from themiddle portion of a fully expanded leaf. The disks were placed on a1.6×4 cm piece of 3M Whatman paper. The paper was folded lengthwise andinserted in a flexible straw. Four hundred micro liters of theextraction buffer (9.5 ml of 0.2 M NaH₂PO₄, 15.5 ml of 0.2 M Na₂HPO₄, 2ml of 0.5 M Na₂EDTA, 100 ml of Triton X100, 1 ml of 10% Sarkosyl, 78 mlof beta-mercaptoethanol, H₂O to bring total volume to 100 ml) waspipetted on to the paper. The straw containing the sample was thenpassed through a rolling device used for squeezing out the extract 1.5mL micro centrifuge tube was placed at the other end of the straw tocollect the extract. The extract was centrifuged for 10 minutes at14,000 rpm in an Eppendorf regrigerated microcentrifuge. The supernatantwas transferred into a new tube. Protein quantitation analysis wasperformed using the standard Bio-Rad Protein Analysis protocol (Bio-RadLaboratories, Hercules, Calif.). The extract was diluted to 2 mg/ml oftotal protein using the extraction buffer.

[0080] For the detection of transgenic protein, Western blot analysiswas performed. Following a standard procedure for protein separation(Laemmli, 1970), 40 μg of protein was loaded in each well of 4-20%gradient polyacrylamide gel (Owl Scientific Co., MA) forelectrophoresis. Subsequently, the protein was transferred onto anitrocellulose membrane using a semi-dry electroblotter (Pharmacia LKBBiotechnology, Piscataway, N.J.). The membrane was incubated for onehour in Blotto (5% milk in TBST solution; 25 mM Tris HCL pH 7.4, 136 mMNaCl, 2.7 mM KCl, 0.1% Tween 20). Thereafter, Blotto was replaced by theprimary antibody solution (in Blotto). After one hour in the primaryantibody, the membrane was washed with TBST for five minutes threetimes. Then the secondary antibody in Blotto (1:2000 dilution of goatanti-rabbit IgG conjugated to horseradish peroxidase; Bio-RadLaboratories). was added to the membrane. After one hour of incubation,the membrane was washed with an excess amount of TBST for 10 minutesfour times. The protein was visualized by using the enhancedchemi-luminescens, ECL method (Amersham, Arlington Heights, Ill.). Thedifferential intensity of the protein bands were measured usingdensitometer (Molecular Dynamics Inc., Sunnyvale, Calif.).

[0081] To determine the expression of TcdA protein in tobaccotransformed with pDAB2041, PAb-EA₀ antibodies were used as the primaryantibodies. The expression levels of TcdA protein varied amongindependent transformation events. The primary plant generated from theevent #2041-13 showed the highest level of pre-pro TcdA expression ofextractable protein. When the leaf pieces from this plant (#2041-13)were used in in vitro propagation, several plants were obtained. Sevenof these plants were analyzed for the expression of the TcdA protein.All but one plant produced the full-length TcdA protein as well as someprocessed peptide components. Using the antibodies specific to Neomycinphospho-transferase, NPT (5 prime-3 prime, Boulder, Colo.), theexpression the selectable marker gene (npt II) was detected. Similarresults were obtained for #2041-29. TABLE 5 Western analysis of plantsderived from event #2041-13. Plant # TcdA NPT (selectable marker)2041-13A + not done 2041-13B + not done 2041-13-1 − + 2041-13-2 + +2041-13-3 + + 2041-13-4 + + 2041-13-5 + +

[0082] C. Nucleic Acid Analysis of Transgenic Tobacco Lines

[0083] Genomic DNA was prepared from a group of 2041 transgenic events.The lines included Magenta box stage 2041-13, and greenhouse stageplants 2041-13-1, 2041-13-2, 2041-13-5, 2041-9, 2041-20A and 2041-20B. Atransgenic GUS line (2023) was included as a negative control. Southernanalysis of these lines was performed. The genomic tobacco DNA wasrestricted with the enzyme SstI which should result in a 8.9 kbhybridization product when hybridized to a tcdA gene specific probe. The8.9 kb hybridization product should consist of the 35T promoter and thetcdA coding region. All 2041 plants contained a band of the expectedsize. Events 2041-9 and -20 appear to be the same line with 5 identicalhybridizing bands. Event 2041-13 produced 6 hybridization fragments withthe tcdA coding region probe. Magenta box and various greenhouse plantsof 2041-13 all produced the same hybridization profile. Thishybridization pattern was different from that of events 2041-9 and -20.

[0084] RNA analysis, using the tcdA coding region probe, was performedon the same group of greenhouse 2041 plants. Immunoblot analysis hadrevealed that plants 2041-9, 2041-20A, 2041-20B, and 2041-13-1 producedno detectable TcdA protein; while 2041-13-2 and 2041-13-5 producedsubstantial amounts of full-length TcdA. Northern analysis was inagreement with the immunoblot result. A faint RNA signal was detectedfor plants 2041-9, 2041-20A, 2041-20B, and 2041-13-1. Only faintlyvisible was a band corresponding to full-length tcdA transcript in plant2041-13.1. In contrast, for plants 2041-13-2 and 2041-13-5 a strong RNAsignal was detected, with a substantial amount of full-length size (˜8.0kb) tcdA transcript. These data support the observed bioassay activityfor this group of plants.

[0085] Genomic DNA was prepared from a second functionally active 2041transgenic event, 2041-29. Southern analysis of this line was performed.A transgenic GUS line (2023) was included as a negative control, DNA ofline 2041-9 was included as a positive control.

[0086] The genomic tobacco DNAs were restricted with the enzyme SstIwhich should result in a 8.9 kb hybridization product when hybridized toa tcdA gene specific probe. The 8.9 kb hybridization product shouldconsist of the 35T promoter and the tcdA coding region. For plant2041-29-5, three hybridization products larger than 8.9 kb the weredetected with the tcdA gene specific probe. Immunoblot analysis hasdemonstrated pre-pro TcdA protein is made by this plant, it is thereforelikely that a restriction site was lost during transformation orregeneration, or the 2041-29 genomic DNA was not thoroughly digested.

[0087] D. Tobacco Leaf-Disk Tests with Tobacco Hornworm ExhibitingInsect Control

[0088] Leaves were sampled from tobacco plants, Nicotiana tabaco,previously transplanted into the greenhouse. A single leaf was sampledfrom each plant on each test date. Leaves were selected from the zonewhere younger elongate leaves transition into older ovate leaves.Excised leaves were placed into 12 oz. cups with the petiole submergedin water to maintain turgor-, and transported to the laboratory.

[0089] Eight, 1.4 cm disks were cut from the center portion of one sideof each leaf (right adaxial side up, with distal portion facing awayfrom the observer). Each disk was placed individually into a well of aC-D International 128 well tray (Pitman, N.J.) into which 0.5 ml of a1.6% aqueous agar solution had been previously pipetted. The solidifiedagar prevented the leaf disks from drying out. The adaxial surface ofthe disk was always oriented up.

[0090] A single neonate tobacco hornworm, Manduca sexta, was placed oneach disk and the wells were sealed with vented plastic lids. The assaywas held at 27° C. and 40% RH. Larval mortality and live-weight datawere collected after 3 days. Data were subjected to analysis of varianceand Duncan's multiple range test (α=0.05)(Proc GLM, SAS Institute Inc.,Cary, N.C.). Data were transformed using a logarithmic function tocorrect a correlation between the magnitude of the mean and variance.TABLE 6 Results of leaf-disk assays from greenhouse grown tobacco plantswith event 2041-13. Weight of Surviving Larvae (mg) & Duncan's Group¹Plant 3 Test TRT Plant Age Pretest Test 1 Test 2 Test 3 Sum. 13non-transformed-2 young — — — 18.8 a* — 14 non-transformed-3 young — — —17.0 ab — 16 non-transformed-5 young — — — 16.4 ab — 32041-13-1(western−) young — 17.6 a 18.2 a 16.1 ab 17.3 a 9 Gus Controlold 19.3 a 14.6 a 16.3 a 14.5 ab 15.1 a 10 non-transformed-1 young — 8.3b 16.8 a 13.9 b 13.0 b 11 2041-20B(western−) old — 10.0 b* 13.7 ab 14.6ab 12.9 b 15 non-transformed-4 young — — — 13.0 bc — 82041-20A(western−) old 15.7 a 8.3 b 11.3 bc 9.2 cd 9.6 c 122041-9(western−) old 19.5 a — — 7.9 d — 7 2041-13-5(western+) young —6.3 bc 9.6 cd 7.2 de 7.7 d 5 2041-13-3(western+) young — 6.4 6.2 e 6.8de** 6.4 de bc**** 1 2041-13A(western+) old 7.2 b 6.8 bc* 7.0 de* 5.4 e6.4 de 6 2041-13-4(western+) young — 4.9 c**** 5.8 e 7.6 d 6.4 de 42041-13-2(western+) young — 5.7 bc 5.7 e** 7.5 d 6.3 de 22041-13B(western +) old — 4.7 c** 5.6 e 7.2 de 5.9 e

[0091] TABLE 7 Results Of Leaf-Disk Assays From Greenhouse Grown TobaccoPlants With Event 2041-29. MEAN WGT (MG)/Duncan's Group Four Test PlantTest 1 Test 2 Test 3 Test 4 Summary 2014-6 GUS 1 15.8 a 16.6a **5.5bc*12.9ab 13.2 a 2014-6 GUS 2 14.4 a *6.6 bc *13.4a 15.2a 12.6 a KY-160NTC 13.4 a 6.7 bc 7.9b 8.5bc 9.1 b 2041-29 4P *4.9 b *7.3b ****6.9b******** 6.3 c 2041-29 7 *5.9 b 5.1bc ***6.7b ***7.2c 6.1 c 2041-29 3P*5.6 b **7.9b *****6.5b ***3.6d 5.9 c 2041-29 2P 6.3 b ****4.7c******4.1c ******4.6d 5.4 c

[0092] All event 2041-29 plants significantly depressed THW larvalweight gain compared to control plants. Average weight depression was49%. Statistically significant mortality occurred in THW larvae exposedto foliage from 2041-29 plants. Mortality averaged 37.5% compared to5.2% in controls.

[0093] E. Isolation and Characterization of Functional PhotorhabdusToxin Protein from Transgenic Plants

[0094] Seven grams of transgenic tobacco plants (2041-13) expressingTcdA (Toxin A) gene were homogenized with 10 ml 50 mM PotassiumPhosphate buffer, pH 7.0 using a bead beater (Biospec Products,Bartlesville, Okla.) according to manufacturer's instructions. Thehomogenate was filtered through four layers of cheese cloth and thencentrifuged at 35,000 g for 15 min. The supernant was collected andfiltered through 0.22 μm Millipore ExpressT™ membrane. It was thenapplied to a Superdex 200 cloumn (2.6×40 cm) which had been equilibratedwith 20 mM Tris buffer, pH 8.0 (Buffer A). The protein was eluted inBuffer A at a flow rate of 3 ml/min. Fractions with 3 ml each werecollected and subjected to southern corn rootworm (SCR) bioassay. It wasfound that fractions corresponding to a native molecular weight around860 kDa had the highest insecticidal activity. Western analysis of theactive fraction using a polyclonal antibody specific to Toxin Aindicated the presence of full-length TcdA peptide. The active fractionswere further combined and applied to a Mono Q 10/10 column which hadbeen equilibrated with Buffer A. Proteins bound to the column were theneluted by a linear gradient of 0 to 1 M NaCl in Buffer A. Fractions with2 ml each were collected and analyzed by both SCR bioassay and Westernusing antibody specific to Toxin A. The results again demonstrated thecorrelation between insecticidal activity and presence of full-lengthTcdA peptide.

[0095] F. Characterization of Progeny Transgenic Plants

[0096] The inheritability of the genetically engineering plantscontaining the Photorhabdus toxin gene was evaluated by generating Flprogeny. Progeny was generated from 2041-13 event by selfing expressionpositive plants. The 2041-13 plants in the greenhouse were allowed toself-pollinate. Seed capsules were collected when mature and wereallowed to dry and after-ripen on the laboratory bench for two weeks.Seed from plant designated 2041-13A was surface-sterilized anddistributed on the surface of medium TOB− without selection, to allowrecovery of nonexpressing or nontransgenic progeny as well as expressingand segregating transgenic siblings. Seed was germinated in a C lightedincubator room (16H light, 28 C). After 1 month, fifty-one seedlings,designated 2041-13A-S1 through S51, were distributed into Magenta boxescontaining medium TOB− to grow further. Three weeks later, leaf samplesfrom these Magenta-box grown seedlings were submitted for evaluation ofthe level of expression of TcdA toxin.

[0097] Leaf samples were tested for kanamycin response by placingsterile leaf segments on medium TOB+ containing 100 mg/L kanamycin inthe light and scoring for tissue growth and color after two weeks. Allleaf pieces showed some positive response, indicating complexsegregation.

[0098] This group of in vitro grown event 2041-13 progeny seedlings wereall transplanted into the greenhouse approximately two months afterseeding onto medium, using the following method. After washing the agarfrom the roots,_plants were transplanted into 5½ inch square pots in asoil mix containing 75% MetroMix and 25% mineral soil. They wereenclosed in a zip-lock bag and plain water added to leave 1-2 inches ofwater in the bottom of the bag after soil absorption. These bags wereclosed and placed under a cart in the greenhouse to protect them fromdirect sunlight. The bags were opened after 5-6 days, and removed after7 days, when the plants were adapted to soil and were moved to the topof the cart for normal greenhouse culture. Plants were ready to test ininsect bioassays at four weeks post transplant.

[0099] F1 progeny were evaluated for expression of protein toxin byimmunological screen and for biological activity by plant bioassays, asdescribed previously, using tobacco hornworm. There existed a positivecorrelation between levels of expression protein toxin and degree ofgrowth inhibition and at higher expression levels mortality wasobserved. The biological activity was observed to be statisticalsignificance with high cofidence levels between populations ofnon-transformed and transformed expressing protein toxin.

[0100] The following table summarizes the results of insect (tobaccohornworm) bioassays conducted with Fl progeny of self-fertilized 2041-13plants genetically engineered to produce the “204” A toxin. The testsincluded 6 non-expressing progeny (protein-negative controls), 45 toxinA expressors, and 4 non-transformed controls (KY-160). Results are fromthree leaf-disk assays (method previously outlined) where eight diskswere used per test. The data were analyzed using analysis of varianceand were blocked by test.

[0101] The treatment effect for each of these analyses indicated thePr>F was less than 0.0001. The Toxin A expressors produced significantcontrol of tobacco hornworm compared to each of the control groups basedon each of the three measures of efficacy. The two control groupsbehaved similarly. Statistical analysis using ANOVA and an LSD test withalpha equal to 0.01 (or 1%) showed differences between the 3 groups. TheLSD test indicated that the non-expressors and the non-transformedplants were similar in larvae weights but the expressors gave weightssignificantly lower than either of the other two groups of plants. Thesedata demonstrated that the genetic basis for insect control wasinheritable and corresponded to the presence of expressed toxin gene.TABLE 8 Tobacco hornworm results from F1 progeny of self- fertilized2041-13 tobacco plants. Mean Value and Duncan's Grouping^(d) TotalWeight Survivor Weight Leaf Area Treatment Group (mg)^(a) (mg)^(b)(cm²)^(c) Non-transformed Control 15.8 a 15.8 a 1.2 a Protein-negativeControl 16.4 a 16.5 a 1.2 a Toxin A Expressor  8.1 b  9.2 b 4.9 b

EXAMPLE 4 Transformation of Maize with a Vector Carrying PlasmidpDAB1834 Encoding Photorhabdus Toxins

[0102] A. Preparation of Maize Transformation Vectors ContainingModified Plant-Optimized tcdA Coding Regions: Plasmid Pdab1834

[0103] Preparation of maize transformation vectors was accomplished intwo steps. First, a modified plant-optimized tcdA coding region wasligated into a plant expression cassette plasmid. In this step, thecoding region was placed under the transcriptional control of a promoterfunctional in maize plant cells. RNA transcription termination andpolyadenylation were mediated by a downstream copy of the terminatorregion from the Agrobacterium nopaline synthase gene. One plasmiddesigned to function in this role is pDAB1538. In the second step, thecomplete gene comprised of the promoter, coding region, and 3′ UTRterminator region was ligated to a plant transformation vector thatcontained a plant expressible selectable marker gene which allowed theselection of transformed maize plant cells amongst a background ofnontransformed cells. An example of such a vector is pDAB367.

[0104] It is a feature of plasmid pDAB1538 that any coding region havingan NcoI site at its 5′ end and a SacI site 3′ to the coding region, whencloned into the unique NcoI and SacI sites of pDAB1538, is placed underthe transcriptional control of the maize ubiquitin1 (ubi1) promoter. Itis also a feature of pDAB1538 that the 5′ untranslated leader (UTR)sequence preceding the NcoI site comprises a polylinker. Additionally itis a feature of pDAB1538 that transcription termination andpolyadenylation of the mRNA containing the introduced coding region aremediated by termination/Poly A addition sequences derived from thenopaline synthase (Nos) gene. Finally, it is a feature of pDAB1538 thatthe entire assembly of promoter/coding region/3′UTR can be obtained as asingle DNA fragment by cleavage at the flanking NotI sites.

[0105] It is a feature of pDAB367 that the phosphinothricin acetyltransferase protein, which has as its substrate phosphinothricin andrelated compounds, is produced in plant cells through transcription ofits coding region mediated by the Cauliflower Mosaic Virus 35S promoterand that termination of transcription plus polyadenylation are mediatedby the nopaline synthase terminator region. It is further a feature ofpDAB367 that any DNA fragment containing flanking NotI sites can becloned into the unique NotI site of pDAB367, thus physically linking theintroduced DNA fragment to the aforementioned selectable marker gene.

[0106] To prepare a maize plant-expressible gene to produce theendoplasmic reticulum-targeted TcdA protein in plant cells, DNA of aplasmid (pA0H_(—)4-ER) containing the plant-optimized, ER-targeted tcdAcoding region, (SEQ ID No:6) was cleaved with restriction enzymes NcoIand SacI, and the large 7610 bp fragment was ligated to similarly-cutDNA of plasmid pDAB1538 to produce plasmid pDAB1832. DNA of pDAB1832 wasthen digested with NotI, and the 9984 bp NotI fragment was ligated intothe unique NotI site of pDAB367 to produce plasmid pDAB1834.

[0107] It is a feature of plasmids pDAB1834 that the ubi1 and 35Spromoters are encoded on the same DNA strand.

[0108] B. Transformation and Regeneration of Transgenic Maize Isolates

[0109] Type II callus cultures were initiated from immature zygoticembryos of the genotype “Hi-II.” (Armstrong et al, (1991) Maize Genet.Coop. Newslett., 65: 92-93). Embryos were isolated from greenhouse-grownears from crosses between Hi-II parent A and Hi-II parent B or F₂embryos derived from a self- or sib-pollination of a Hi-II plant.Immature embryos (1.5 to 3.5 mm) were cultured on initiation mediumconsisting of N6 salts and vitamins (Chu et al, (1978) The N6 medium andits application to anther culture of cereal crops. Proc. Symp. PlantTissue Culture, Peking Press, 43-56), 1.0 mg/L 2,4-D, 25 mM L-proline,100 mg/L casein hydrolysate, 10 mg/L AgNO₃, 2.5 g/L GELRITE(Schweizerhall, South Plainfield, N.J.), and 20 g/L sucrose, with a pHof 5.8. After four to six weeks callus was subcultured onto maintenancemedium (initiation medium in which AgNO₃ was omitted and L-proline wasreduced to 6 mM). Selection for Type II callus took place for ca. 12-16weeks.

[0110] Plasmid pDAB1834 was transformed into embryogenic callus. Forblasting, 140 μg of plasmid DNA was precipitated onto 60 mg ofalcohol-rinsed, spherical gold particles (1.5-3.0 μm diameter, AldrichChemical Co., Inc., Milwaukee, Wis.) by adding 74 μL of 2.5M CaCl₂H₂Oand 30 μL of 0.1M spermidine (free base) to 300 μL of plasmid DNA andH₂O. The solution was immediately vortexed and the DNA-coated goldparticles were allowed to settle. The resulting clear supernatant wasremoved and the gold particles were resuspended in 1 ml of absoluteethanol. This suspension was diluted with absolute ethanol to obtain 15mg DNA-coated gold/mL.

[0111] Approximately 600 mg of embryogenic callus tissue was spread overthe surface of Type II callus maintenance medium as described hereinlacking casein hydrolysate and L-proline, but supplemented with 0.2 Msorbitol and 0.2 M mannitol as an osmoticum. Following a 4 hpre-treatment, tissue was transferred to culture dishes containingblasting medium (osmotic media solidified with 20 g/L TC agar(PhytoTechnology Laboratories, LLC, Shawnee Mission, Kans.) instead of 7g/L GELRITE. Helium blasting accelerated suspended DNA-coated goldparticles towards and into the prepared tissue targets. The device usedwas an earlier prototype of that described in U.S. Pat. No. 5,141,131which is incorporated herein by reference. Tissues were covered with astainless steel screen (104 μm openings)and placed under a partialvacuum of 25 inches of Hg in the device chamber. The DNA-coated goldparticles were further diluted 1:1 with absolute ethanol prior toblasting and were accelerated at the callus targets four times using ahelium pressure of 1500 psi, with each blast delivering 20 μL of theDNA/gold suspension. Immediately post-blasting, the tissue wastransferred to osmotic media for a 16-24 h recovery period. Afterwards,the tissue was divided into small pieces and transferred to selectionmedium (maintenance medium lacking casein hydrolysate and L-proline butcontaining 30 mg/L BASTA® (AgrEvo, Berlin, Germany)). Every four weeksfor 3 months, tissue pieces were non-selectively transferred to freshselection medium. After 7 weeks and up to 22 weeks, callus sectors foundproliferating against a background of growth-inhibited tissue wereremoved and isolated. The resulting BASTA®-resistant tissue wassubcultured biweekly onto fresh selection medium. Following westernanalysis, positive transgenic lines were identified and transferred toregeneration media. Western-negative lines underwent subsequent RNA spotblot analysis to identify negative controls for regeneration.

[0112] Regeneration was initiated by transferring callus tissue tocytokinin-based induction medium, which consisted of Murashige and Skoogsalts, hereinafter MS salts, and vitamins (Murashige and Skoog, (1962)Physiol. Plant. 15: 473-497) 30 g/L sucrose, 100 mg/L myo-inositol, 30g/L mannitol, 5 mg/L 6-benzylaminopurine, hereinafter BAP, 0.025 mg/L2,4-D, 30 mg/L BASTA®, and 2.5 g/L GELRITE at pH 5.7. The cultures wereplaced in low light (125 ft-candles) for one week followed by one weekin high light (325 ft-candles). Following a two week induction period,tissue was non-selectively transferred to hormone-free regenerationmedium, which was identical to the induction medium except that itlacked 2,4-D and BAP, and was kept in high light. Small (1.5-3 cm)plantlets were removed and placed in 150×25 mm culture tubes containingSH medium (SH salts and vitamins (Schenk and Hildebrandt, (1972) Can. J.Bot. 50:199-204), 10 g/L sucrose, 100 mg/L myo-inositol, 5 mL/L FeEDTA,and 2.5 g/L GELRITE, pH 5.8). Plantlets were transferred to 12 cm potscontaining approximately 0.25 kg of METRO-MIX 360 (The Scotts Co.Marysville, Ohio) in the greenhouse as soon as they exhibited growth anddeveloped a sufficient root system. They were grown with a 16 hphotoperiod supplemented by a combination of high pressure sodium andmetal halide lamps, and were watered as needed with a combination ofthree independent Peters Excel fertilizer formulations (Grace-SierraHorticultural Products Company, Milpitas, Calif.). At the 6-8 leafstage, plants were transplanted to five gallon pots containingapproximately 4 kg METRO-MIX 360, and grown to maturity.

EXAMPLE 5 Characterization Of Transgenic Maize Plants ExpressingPhotorhabdus Toxin That Confer Insect Control

[0113] A. Insect Bioassays

[0114] A single leaf was sampled from each plant in each test. Eight,1.4 cm disks were cut from the outer portion of each leaf (approximately30 cm long) avoiding the center vein. Each disk was placed individuallyinto a well of a C-D International 128 well tray (Pitman, N.J.) intowhich 0.5 ml of a 1.6% aqueous agar solution had been previouslypipetted. The solidified agar prevented the leaf disks from drying out.The adaxial surface of the disk was always oriented up.

[0115] Five neonate southern corn rootworms, Diabrotica undecimpunctatahowardi, were placed on each disk and the wells were sealed with ventedplastic lids. The assay was held at 27° C. and 40% RH. Larval mortalityand live-weight data were collected after 3 days. Data were subjected toanalysis of variance and Duncan's multiple range test (α=0.05)(Proc GLM,SAS Institute Inc., Cary, N.C.). Weight data were transformed using alogarithmic function to correct a correlation between the magnitude ofthe mean and variance. TABLE 9 Results of Maize Leaf-disk Test vs SCRMean Survival Mean % Kill Weight (mg) Treatment (Duncan's) (Duncan's)1834 - 11 68 A 0.064 A 1834 - 17 44 B 0.093 B 1834 - 15 26 BC 0.127 CHiII control 13 C 0.161 C

[0116] The results shown in Table 9 demonstrated that two eventsexpressing TcdA protein were statistically distinct from control linesbioassayed using SCR neonates by mortality and survival weight criteria.These results demonstrated that southern corn rootworm were functionallyeffected by feeding on maize plants containing and expressing the tcdAgene. Those plants from 1834-11 were used to generate progeny fortesting of inheritability of transgene.

[0117] B. Production and Progeny Test of tcdA Transgenic Maize

[0118] Origin and growth of progeny plants: Sibling plants 1834-11-07and 1834-11-08, clonally derived by regeneration from the callus oftransgenic maize event 1834-11, were transplanted to the greenhouse andpollinated with inbred OQ414. Seeds obtained from these crosses,comprising seed lots 1834-11-07A and 1834-11-08A, were planted inRootrainers (1½ inch×2 inch×8 inch deep, product #647, C. Hummert Intl.,Earth City, Mo.) filled with Metro-Mix 360 soilless mix (ScottsTerra-Lite, available from Hummert Intl.) and top irrigated withHoagland's nutrient solution. (Hoagland's solution contains 229 ppmnitrogen as nitrate, 24.6 ppm nitrogen as ammonium, 26 ppm P, 157 ppm K,187 ppm Ca, 49 ppm Mg. and 30 ppm Na.)

[0119] Greenhouse conditions for this trial were: 16 hour days, daylightsupplemented by metal halide lamps as needed to achieve a minimum of 600?Einsteins/cm² PAR, and ambient temperature 30 C days, 22 C nights.

[0120] Leaves were sampled for protein determination approximately oneweek after planting. Leaf bioassays were conducted 2-3 weeks afterplanting; root bioassays were initiated approximately 3 weeks postplanting.

[0121] Protein analysis of progeny plants: Protein was extracted fromleaf and root samples harvested from transgenic plants, line 1834-11progenies, and non-transformed plants. Each sample was placed on a 1.6×4cm piece of 3M Whatman T paper. The paper was folded lengthwise andinserted in a flexible straw. A volume of 350 μl of an extraction buffer(9.5 ml of 0.2 M NaH₂PO₄, 15.5 ml of 0.2 M Na₂HPO₄, 2 ml of 0.5 MNa₂EDTA, 100 ml of Triton X-100, 1 ml of 10% Sarkosyl, 78 ml ofbeta-mercaptoethanol, H₂O to bring total volume to 100 ml, 50 μg/mlAntipain, 50 μg/ml Leupeptin, 0.1 mM Chymostatin, 5 μg/ml Pepstatin) waspipetted on to the paper. The straw containing the sample was thenpassed through a rolling device used for squeezing the extract into a1.5 ml microcentrifuge tube. The extract was centrifuged for 10 minutesat 14,000 rpm in an Eppendorf refrigerated micro-centrifuge. Thesupernatant was transferred into a new tube. The amount of the totalextractable protein was determined using a standard BioRad ProteinAnalysis protocol (BioRad Laboratories, Hercules, Calif.).

[0122] The presence of the TcdA protein was visualized by Western blotanalysis following a standard procedure for protein separation (Laemmli,1970). A volume of twenty μl of extract was loaded in each well of 4-20%gradient polyacrylamide gel (Owl Scientific Co., MA) forelectrophoresis. Subsequently, the protein was transferred onto anitrocellulose membrane using a semi-dry electroblotter (Pharmacia LKBBiotechnology, Piscataway, N.J.). The membrane was incubated for onehour in TBST-M solution (10% milk in TBST solution; 25 mM Tris HCL pH7.4, 136 mM NaCl, 2.7 mM KCl, 0.1% Tween 20). Thereafter, the primaryantibody (Anti-TcdA in TBST-M) was added. After one hour, the membranewas washed with TBST for five minutes, three times. Then the secondaryantibody solution (goat anti-rabbit IgG conjugated to horseradishperoxidase; Bio-Rad Laboratories, in TBST-M) was added to the membrane.After one hour of incubation, the membrane was washed with an excessamount of TBST for 10 minutes, four times. The protein was visualizedusing the Super Signal® West Pico chemiluminescence method (PierceChemical Co., Rockford, Ill.). The protein blot was exposed on aHyper-film (Amersham, Arlington Heights, Ill.) and was developed within3 minutes. The intensity of the protein band was measured using adensitometer (Molecular Dynamics Inc., Sunnyvale, Calif.) and comparedto standards.

[0123] Three of six plants from seed lot 1834-11-07A and three of sixplants from seed lot 1834-11-08A produced detectable levels of TcdAprotein (Table 1). Approximately 3.8 to 13.3 ppm of TcdA were detectedin the leaf blades and 4.1 to 8.4 ppm were detected in the leaf tips ofthe protein-positive plants. The amounts of TcdA protein detected in theroots were slightly lower than those found in the leaves.

[0124] Insect bioassays with progeny plants: Plants were selected forbioassay based on results from Western blot analysis. Twelve (12), 6.4mm diameter leaf discs were cut from the youngest leaf of each 2 weekold seedling. Each disc was placed in a well of a 128-well tray (CDInternational) containing approximately 0.5 mL of a solidified 2% agarin water solution. Two neonate southern corn rootworm, Diabroticaundecimpunctata howardi (Barber) (SCR), were placed in each well with aleaf disc. Trays were covered with perforated lids and maintained undera controlled environment for 3 days (28 C; 16 hours light:8 hours dark;approx. 60% relative humidity). Living larvae from 4 leaf discs werepooled and weighed producing 3 weight determinations per plant. Averageweights were calculated by dividing the pooled weight by the number ofsurvivors. Differences in average weights of SCR fed leaf discs fromprotein positive and protein negative plants were assessed usinganalysis of variance on the natural log-transformed average weights(Minitab, v. 12.2, Minitab Inc., State College, Pa.).

[0125] Root bioassays were initiated approximately 1 week after theinitiation of the leaf disc bioassays. Approximately 24 h prior toeclosion, SCR eggs were suspended in a 0.15% solution of agar in waterto a concentration of 100 eggs/ml. Plants were inoculated with SCR eggsby pipetting 2.0 ml of the egg suspension (ie., approximately 200 eggs)just below the soil surface at the base of each plant. Two weeks afterinoculation, plants were removed from their Rootrainer pots, their rootswashed free of potting mix, and scored for rootworm damage based on a 1(resistant) to 9 (susceptible) rating system (Welch, 1977). The resultsof the root ratings were examined using non-parametric tests todetermine if the distribution of root ratings from the protein positiveplants was the same as the distribution of the ratings from the proteinnegative plants. Testing was done at the 5% significance level.(StatXact v.3, CYTEL Software Corporation, Cambridge Mass.)

[0126] Results from leaf and root bioassays of tcdA protein positive andprotein negative progeny plants are summarized in Table 10. The averageweights of SCR larvae fed leaf discs from protein positive plants weresignificantly lower than those of larvae fed leaf discs from proteinnegative plants (F=4.6; d.f.=1, 34; P≦0.001. The Kolmogorov-Smirnov 2sample test (p=0.04) and the Wald Wolfowitz runs test (p=0.001)indicated that the protein positive and protein negative root ratingdistributions were not similar. The Wilcoxon-Mann-Whitney test(p=0.0206) and the Normal Scores test (p=0.206) indicated that theaverage score for the protein positive plants was lower than the averageroot rating from the protein negative plants. TABLE 10 Protein analysisand insect bioassay results with progeny of TcdA transgenic maize. LeafDisc Root Bioassay Plant TcdA Bioassay Root Rating Number Protein Avg.Wt. (mg) (1-9) 1834-11-07A-30 PRO− 0.190 8 1834-11-08A-21 PRO− 0.196 91834-11-08A-16 PRO− 0.195 9 1834-11-08A-14 PRO− 0.137 9 1834-11-07A-22PRO− 0.208 9 1834-11-07A-20 PRO− 0.175 9 1834-11-07A-26 PRO+ 0.118 91834-11-08A-17 PRO+ 0.132 8 1834-11-07A-14 PRO+ 0.110 2 1834-11-07A-11PRO+ 0.106 4 1834-11-08A-28 PRO+ 0.129 8 1834-11-08A-27 PRO+ 0.108 4

[0127] DNA analysis of progeny plants: Leaf samples from 1834-11.7A and1834-11.8A progeny plants were in conical 50 ml polypropylene tubes anddried in a Labconco Freeze Dry Lyophilizer (Kansas City, Mo.) for 1-2days. Lyophilized leaves were then ground in a Tecator Cyclotec 1093Sample mill grinder (Hoganas, Sweden) and stored at −20C. Genomic DNAwas extracted by the following procedure: (1) to a 25 ml Conical tubecontaining 300-500 mg of ground tissue, 9 ml of CTAB (cetyltrimethylammonium bromide solution) was added, and incubated at 65° C.for 1 hour; (2) 4.5 ml of chloroform: octanol (24:1) was added and mixedgently for 5 minutes; (3) samples were centrifuged at 2000 rpm and DNAwas precipitated from the supernatant with an equal volume ofisopropanol; (4) DNA was collected on a glass hook, washed in ethanol,and dissolved in TE (10 mM Tris.HCl, 0.5 mM EDTA, pH 8.0).

[0128] Genomic DNA was digested at 37° C. for 2 hours in an Eppendorftube containing the following mixture: 8 μl of 800 ug/ml DNA, 2 μl 1mg/ml BSA (Bovine serum albumin),2 μl 10× buffer, 1 μl SacI, 1 μl EcoRI,and 6 μl H₂O. Digested DNA samples were electrophoresed overnight at 40mA in a 0.85% SeaKem LE agarose gel(FMC, Rockland, Me.). The gel wasblotted onto Millipore Immobilon-Ny+ (Bedford, Mass.) membrane overnightin 20×SSC (NaCl 175.2 g/l, Na citrate 88 g/l). The probe DNA was cutwith BamHI/SacI (NEB, Beverly, Mass.) from pDAB1551 plasmid, whichreleased a 7356 bp fragment containing the open reading frame of therebuilt tcdA gene. This 7356 bp fragment was labeled with P32 using aStratagene Prime-it RmT dCTP-Labeling Reactions kit (La Jolla, Calif.)and used for Southern hybridization. Hybridization was conducted inhybridization buffer (10% polyethylene glycol, 7% SDS [Sodium dodecylsulfate], 0.6×SSC, 10 mM NaPO₄, 5 mM EDTA, 10 μg/ml denatured salmonsperm) at 60° C. overnight. After hybridization, the membrane was washedwith 10×SSC plus 0.1% SDS at 60° C. for 30 min and exposed to X ray film(Hyperfilm® MP, Amershan Life Sciences, Piscataway, N.J.) for 1-2 days.

[0129] Results summarized indicate that a pattern of 8 hybridizing bands(the size of the expected fragment and larger) cosegregated with proteinexpression in 50% of all progeny assayed. These results arecharacteristic of a complex insertion at a single site. All seedlingscontaining the insert also expressed toxin protein.

EXAMPLE 6 Transformation of Rice with a Vector Carrying Plasmid pDAB1553Encoding Photorhabdus Toxins

[0130] A. Plasmid pDAB1553

[0131] Plasmid pDAB1553 containing tcdA driven by the maize ubiquitinlpromoter and hpt (hygromycin phosphotransferase providing resistance tothe antibiotic hygromycin) under the control of 35T (a modified 35Spromoter), was used for transformation.

[0132] Preparation of rice transformation vectors was accomplished intwo steps. First, a modified plant-optimized tcdA coding region wasligated into a rice plant expression cassette plasmid. In this step, thecoding region was placed under the transcriptional control of a promoterfunctional in plant cells. RNA transcription termination andpolyadenylation were mediated by a downstream copy of the terminatorregion from the Agrobacterium nopaline synthase gene. One plasmiddesigned to function in this role is plasmid pDAB1538 (described in thesection on maize transformation vectors). In the second step, thecomplete gene comprised of the promoter, coding region, and terminatorregion was ligated to a rice plant transformation vector that containeda plant expressible selectable marker gene which allowed the selectionof transformed rice plant cells amongst a background of nontransformedcells. An example of such a vector is pDAB354-Not1.

[0133] It is a feature of pDAB354-Not1 that the hygromycinphosphotransferase protein, which has as its substrate hygromycin B andrelated compounds, is produced in plant cells through transcription ofits coding region mediated by the Cauliflower Mosaic Virus 35S promoterand that termination of transcription plus polyadenylation are mediatedby the nopaline synthase terminator region. It is further a feature ofpDAB354-Not1 that any DNA fragment containing flanking NotI sites can becloned into the unique NotI site of pDAB354-Not1, thus physicallylinking the introduced DNA fragment to the aforementioned selectablemarker gene.

[0134] To prepare a plant-expressible gene to produce the non-targetedTcdA protein in rice plant cells, DNA of a plasmid (pAOH_(—)4-OPTI)containing the plant-optimized tcdA coding region, (SEQ ID No:3) wascleaved with restriction enzymes NcoI and SacI, and the large 7550 bpfragment was ligated to similarly-cut DNA of plasmid pDAB1538 to produceplasmid pDAB1551. DNA of pDAB1551 was then digested with NotI, and thelarge 9933 bp fragment was ligated to NotI digested DNA of pDAB354-NotIto produce plasmid pDAB1553.

[0135] It is a feature of plasmid pDAB1553 that the ubil and 35Spromoters are encoded on the same DNA strand.

[0136] B. Production of Rice Transgenics

[0137] For initiation of embryogenic callus, mature seeds of a Japonicacultivar, Taipei 309 were dehusked and surface-sterilized in 70% ethanolfor 2-5 min. followed by a 30-45 min soak in 50% commercial bleach (2.6%sodium hypochlorite) with a few drops of ‘Liquinox’ soap. The seeds werethen rinsed 3 times in sterile distilled water and placed on filterpaper before transferring to ‘callus induction’ medium (i.e., NB). TheNB medium consisted of N6 macro elements (Chu, 1978, The N6 medium andits application to anther culture of cereal crops. Proc. Symp. PlantTissue Culture, Peking Press, p43-56), B5 micro elements and vitamins(Gamborg et al., 1968, Nutrient requirements of suspension cultures ofsoybean root cells. Exp. Cell Res. 50: 151-158), 300 mg/L caseinhydrolysate, 500 mg/L L-proline, 500 mg/L L-glutamine, 30 g/L sucrose, 2mg/L 2,4-dichloro-phenoxyacetic acid (2,4-D), and 2.5 g/L gelrite(Schweizerhall, N.J.) with the pH adjusted to 5.8. The mature seedcultured on ‘induction’ media were incubated in the dark at 28° C. After3 weeks of culture, the emerging primary callus induced from thescutellar region of mature embryo was transferred to fresh NB medium forfurther maintenance.

[0138] About 140 μg of plasmid pDAB1553 DNA was precipitated onto 60 mgof 1.0 micron (Bio-Rad) gold particles as described herein.

[0139] For helium blasting, actively growing embryogenic calluscultures, 2-4 mm in size, were subjected to a high osmoticum treatment.This treatment included placing of callus on NB medium with 0.2 Mmannitol and 0.2 M sorbitol (Vain et al., 1993, Osmoticum treatmentenhances particle bombardment-mediated transient and stabletransformation of maize. Plant Cell Rep. 12: 84-88) for 4 h beforehelium blasting. Following osmoticum treatment, callus cultures weretransferred to ‘blasting’ medium (NB+2% agar) and covered with astainless steel screen (230 micron). The callus cultures were blasted at2,000 psi helium pressures twice per target. After blasting, callus wastransferred back to the media with high osmoticum overnight beforeplacing on selection medium, which consisted NB medium with 30 mg/Lhygromycin. After 2 weeks, the cultures were transferred to freshselection medium with a higher concentration of selection agent, i.e.,NB+50 mg/L hygromycin (Li et al., 1993, An improved rice transformationsystem using the biolistic method. Plant Cell Rep. 12: 250-255).

[0140] Compact, white-yellow, embryogenic callus cultures, recovered onNB+50 mg/L hygromycin, were regenerated by transferring to‘pre-regeneration’ (PR) medium+50 mg/L hygromycin. The PR mediumconsisted of NB medium with 2 mg/L benzyl aminopurine (BAP), 1 mg/Lnaphthalene acetic acid (NAA), and 5 mg/L abscisic acid (ABA). After 2weeks of culture in the dark, they were transferred to ‘regeneration’(RN) medium. The composition of RN medium is NB medium with 3 mg/L BAP,and 0.5 mg/L NAA. The cultures on RN medium were incubated for 2 weeksat 28° C. under high fluorescent light (325-ft-candles). The plantletswith 2 cm shoot were transferred to ½ MS medium (Murashige and Skoog,1962, A revised medium for rapid growth and bioassays with tobaccotissue cultures. Physiol. Plant.15:473-497) with ½ B5 vitamins, 10 g/Lsucrose, 0.05 mg/L NAA, 50 mg/L hygromycin and 2.5 g/L gelrite adjustedto pH 5.8 in magenta boxes. When plantlets were established withwell-developed root systems, they were transferred to soil (1 metromix:1 top soil) and raised in the greenhouse (29/24° C. day/night cycle,50-60% humidity, 12 h photoperiod) until maturity.

EXAMPLE 7

[0141] Chacterization of Transgenic Rice Plants Expressing PhotorhabdusToxin that Confer Insect Control.

[0142] A. Insect Bioassays

[0143] Insect bioassays were performed using leaf discs and shown to behighly effective in controlling Southern corn rootworm. Diabroticaundecimpunctata howardi eggs are obtained from French Ag Research andhatched in petri dishes held at 28.5° C. and 40% RH. The aerial partsare sampled from the transgenic plants and placed, singly into invertedpetri dishes (100×15 mm) containing 15 ml of 1.6% aqueous agar in thebottom to provide humidity and filter paper in the top to absorbcondensation. These preparations are infested with five neonate larvaeper dish and held at 28.5° C. and 40% RH for 3 days. Mortality andlarval weights are recorded. Weight data were transformed using alogarithmic function to correct a correlation between the magnitude ofthe mean and variance. TABLE 11 Average Survivor Presence TcdA Weight inmg¹ greenhouse-grown (Duncan's plants (number of +/ Treatment Grouping)number of plants tested) GUS 0.390 A − Control 1553-33 0.170 BCD ++1553-44 0.167 BCD +++ 1553-62 0.125 CD +++ 1553-41 0.100 D +++

[0144] Insect groups weighing less than 0.1 mg were set to 0.03 mginstead of zero to conduct a more conservative analysis. Weight datawere transformed (Log10) for analyses. A single replicate was used oneach of three test dates. Plants were sampled from magenta boxes.

[0145] The results demonstrate that in leaf disc bioassays, several riceevents derived by transformation with tcdA gene were demonstrated tostatistically have a functional affect on corn rootworm neonate.

1 6 1 7551 DNA Photorhabdus luminescens CDS (1)..(7548) 1 atg aac gagtct gta aaa gag ata cct gat gta tta aaa agc cag tgt 48 Met Asn Glu SerVal Lys Glu Ile Pro Asp Val Leu Lys Ser Gln Cys 1 5 10 15 ggt ttt aattgt ctg aca gat att agc cac agc tct ttt aat gaa ttt 96 Gly Phe Asn CysLeu Thr Asp Ile Ser His Ser Ser Phe Asn Glu Phe 20 25 30 cgc cag caa gtatct gag cac ctc tcc tgg tcc gaa aca cac gac tta 144 Arg Gln Gln Val SerGlu His Leu Ser Trp Ser Glu Thr His Asp Leu 35 40 45 tat cat gat gca caacag gca caa aag gat aat cgc ctg tat gaa gcg 192 Tyr His Asp Ala Gln GlnAla Gln Lys Asp Asn Arg Leu Tyr Glu Ala 50 55 60 cgt att ctc aaa cgc gccaat ccc caa tta caa aat gcg gtg cat ctt 240 Arg Ile Leu Lys Arg Ala AsnPro Gln Leu Gln Asn Ala Val His Leu 65 70 75 80 gcc att ctc gct ccc aatgct gaa ctg ata ggc tat aac aat caa ttt 288 Ala Ile Leu Ala Pro Asn AlaGlu Leu Ile Gly Tyr Asn Asn Gln Phe 85 90 95 agc ggt aga gcc agt caa tatgtt gcg ccg ggt acc gtt tct tcc atg 336 Ser Gly Arg Ala Ser Gln Tyr ValAla Pro Gly Thr Val Ser Ser Met 100 105 110 ttc tcc ccc gcc gct tat ttgact gaa ctt tat cgt gaa gca cgc aat 384 Phe Ser Pro Ala Ala Tyr Leu ThrGlu Leu Tyr Arg Glu Ala Arg Asn 115 120 125 tta cac gca agt gac tcc gtttat tat ctg gat acc cgc cgc cca gat 432 Leu His Ala Ser Asp Ser Val TyrTyr Leu Asp Thr Arg Arg Pro Asp 130 135 140 ctc aaa tca atg gcg ctc agtcag caa aat atg gat ata gaa tta tcc 480 Leu Lys Ser Met Ala Leu Ser GlnGln Asn Met Asp Ile Glu Leu Ser 145 150 155 160 aca ctc tct ttg tcc aatgag ctg tta ttg gaa agc att aaa act gaa 528 Thr Leu Ser Leu Ser Asn GluLeu Leu Leu Glu Ser Ile Lys Thr Glu 165 170 175 tct aaa ctg gaa aac tatact aaa gtg atg gaa atg ctc tcc act ttc 576 Ser Lys Leu Glu Asn Tyr ThrLys Val Met Glu Met Leu Ser Thr Phe 180 185 190 cgt cct tcc ggc gca acgcct tat cat gat gct tat gaa aat gtg cgt 624 Arg Pro Ser Gly Ala Thr ProTyr His Asp Ala Tyr Glu Asn Val Arg 195 200 205 gaa gtt atc cag cta caagat cct gga ctt gag caa ctc aat gca tca 672 Glu Val Ile Gln Leu Gln AspPro Gly Leu Glu Gln Leu Asn Ala Ser 210 215 220 ccg gca att gcc ggg ttgatg cat caa gcc tcc cta ttg ggt att aac 720 Pro Ala Ile Ala Gly Leu MetHis Gln Ala Ser Leu Leu Gly Ile Asn 225 230 235 240 gct tca atc tcg cctgag cta ttt aat att ctg acg gag gag att acc 768 Ala Ser Ile Ser Pro GluLeu Phe Asn Ile Leu Thr Glu Glu Ile Thr 245 250 255 gaa ggt aat gct gaggaa ctt tat aag aaa aat ttt ggt aat atc gaa 816 Glu Gly Asn Ala Glu GluLeu Tyr Lys Lys Asn Phe Gly Asn Ile Glu 260 265 270 ccg gcc tca ttg gctatg ccg gaa tac ctt aaa cgt tat tat aat tta 864 Pro Ala Ser Leu Ala MetPro Glu Tyr Leu Lys Arg Tyr Tyr Asn Leu 275 280 285 agc gat gaa gaa cttagt cag ttt att ggt aaa gcc agc aat ttt ggt 912 Ser Asp Glu Glu Leu SerGln Phe Ile Gly Lys Ala Ser Asn Phe Gly 290 295 300 caa cag gaa tat agtaat aac caa ctt att act ccg gta gtc aac agc 960 Gln Gln Glu Tyr Ser AsnAsn Gln Leu Ile Thr Pro Val Val Asn Ser 305 310 315 320 agt gat ggc acggtt aag gta tat cgg atc acc cgc gaa tat aca acc 1008 Ser Asp Gly Thr ValLys Val Tyr Arg Ile Thr Arg Glu Tyr Thr Thr 325 330 335 aat gct tat caaatg gat gtg gag cta ttt ccc ttc ggt ggt gag aat 1056 Asn Ala Tyr Gln MetAsp Val Glu Leu Phe Pro Phe Gly Gly Glu Asn 340 345 350 tat cgg tta gattat aaa ttc aaa aat ttt tat aat gcc tct tat tta 1104 Tyr Arg Leu Asp TyrLys Phe Lys Asn Phe Tyr Asn Ala Ser Tyr Leu 355 360 365 tcc atc aag ttaaat gat aaa aga gaa ctt gtt cga act gaa ggc gct 1152 Ser Ile Lys Leu AsnAsp Lys Arg Glu Leu Val Arg Thr Glu Gly Ala 370 375 380 cct caa gtc aatata gaa tac tcc gca aat atc aca tta aat acc gct 1200 Pro Gln Val Asn IleGlu Tyr Ser Ala Asn Ile Thr Leu Asn Thr Ala 385 390 395 400 gat atc agtcaa cct ttt gaa att ggc ctg aca cga gta ctt cct tcc 1248 Asp Ile Ser GlnPro Phe Glu Ile Gly Leu Thr Arg Val Leu Pro Ser 405 410 415 ggt tct tgggca tat gcc gcc gca aaa ttt acc gtt gaa gag tat aac 1296 Gly Ser Trp AlaTyr Ala Ala Ala Lys Phe Thr Val Glu Glu Tyr Asn 420 425 430 caa tac tctttt ctg cta aaa ctt aac aag gct att cgt cta tca cgt 1344 Gln Tyr Ser PheLeu Leu Lys Leu Asn Lys Ala Ile Arg Leu Ser Arg 435 440 445 gcg aca gaattg tca ccc acg att ctg gaa ggc att gtg cgc agt gtt 1392 Ala Thr Glu LeuSer Pro Thr Ile Leu Glu Gly Ile Val Arg Ser Val 450 455 460 aat cta caactg gat atc aac aca gac gta tta ggt aaa gtt ttt ctg 1440 Asn Leu Gln LeuAsp Ile Asn Thr Asp Val Leu Gly Lys Val Phe Leu 465 470 475 480 act aaatat tat atg cag cgt tat gct att cat gct gaa act gcc ctg 1488 Thr Lys TyrTyr Met Gln Arg Tyr Ala Ile His Ala Glu Thr Ala Leu 485 490 495 ata ctatgc aac gcg cct att tca caa cgt tca tat gat aat caa cct 1536 Ile Leu CysAsn Ala Pro Ile Ser Gln Arg Ser Tyr Asp Asn Gln Pro 500 505 510 agc caattt gat cgc ctg ttt aat acg cca tta ctg aac gga caa tat 1584 Ser Gln PheAsp Arg Leu Phe Asn Thr Pro Leu Leu Asn Gly Gln Tyr 515 520 525 ttt tctacc ggc gat gag gag att gat tta aat tca ggt agc acc ggc 1632 Phe Ser ThrGly Asp Glu Glu Ile Asp Leu Asn Ser Gly Ser Thr Gly 530 535 540 gat tggcga aaa acc ata ctt aag cgt gca ttt aat att gat gat gtc 1680 Asp Trp ArgLys Thr Ile Leu Lys Arg Ala Phe Asn Ile Asp Asp Val 545 550 555 560 tcgctc ttc cgc ctg ctt aaa att acc gac cat gat aat aaa gat gga 1728 Ser LeuPhe Arg Leu Leu Lys Ile Thr Asp His Asp Asn Lys Asp Gly 565 570 575 aaaatt aaa aat aac cta aag aat ctt tcc aat tta tat att gga aaa 1776 Lys IleLys Asn Asn Leu Lys Asn Leu Ser Asn Leu Tyr Ile Gly Lys 580 585 590 ttactg gca gat att cat caa tta acc att gat gaa ctg gat tta tta 1824 Leu LeuAla Asp Ile His Gln Leu Thr Ile Asp Glu Leu Asp Leu Leu 595 600 605 ctgatt gcc gta ggt gaa gga aaa act aat tta tcc gct atc agt gat 1872 Leu IleAla Val Gly Glu Gly Lys Thr Asn Leu Ser Ala Ile Ser Asp 610 615 620 aagcaa ttg gct acc ctg atc aga aaa ctc aat act att acc agc tgg 1920 Lys GlnLeu Ala Thr Leu Ile Arg Lys Leu Asn Thr Ile Thr Ser Trp 625 630 635 640cta cat aca cag aag tgg agt gta ttc cag cta ttt atc atg acc tcc 1968 LeuHis Thr Gln Lys Trp Ser Val Phe Gln Leu Phe Ile Met Thr Ser 645 650 655acc agc tat aac aaa acg cta acg cct gaa att aag aat ttg ctg gat 2016 ThrSer Tyr Asn Lys Thr Leu Thr Pro Glu Ile Lys Asn Leu Leu Asp 660 665 670acc gtc tac cac ggt tta caa ggt ttt gat aaa gac aaa gca gat ttg 2064 ThrVal Tyr His Gly Leu Gln Gly Phe Asp Lys Asp Lys Ala Asp Leu 675 680 685cta cat gtc atg gcg ccc tat att gcg gcc acc ttg caa tta tca tcg 2112 LeuHis Val Met Ala Pro Tyr Ile Ala Ala Thr Leu Gln Leu Ser Ser 690 695 700gaa aat gtc gcc cac tcg gta ctc ctt tgg gca gat aag tta cag ccc 2160 GluAsn Val Ala His Ser Val Leu Leu Trp Ala Asp Lys Leu Gln Pro 705 710 715720 ggc gac ggc gca atg aca gca gaa aaa ttc tgg gac tgg ttg aat act 2208Gly Asp Gly Ala Met Thr Ala Glu Lys Phe Trp Asp Trp Leu Asn Thr 725 730735 aag tat acg ccg ggt tca tcg gaa gcc gta gaa acg cag gaa cat atc 2256Lys Tyr Thr Pro Gly Ser Ser Glu Ala Val Glu Thr Gln Glu His Ile 740 745750 gtt cag tat tgt cag gct ctg gca caa ttg gaa atg gtt tac cat tcc 2304Val Gln Tyr Cys Gln Ala Leu Ala Gln Leu Glu Met Val Tyr His Ser 755 760765 acc ggc atc aac gaa aac gcc ttc cgt cta ttt gtg aca aaa cca gag 2352Thr Gly Ile Asn Glu Asn Ala Phe Arg Leu Phe Val Thr Lys Pro Glu 770 775780 atg ttt ggc gct gca act gga gca gcg ccc gcg cat gat gcc ctt tca 2400Met Phe Gly Ala Ala Thr Gly Ala Ala Pro Ala His Asp Ala Leu Ser 785 790795 800 ctg att atg ctg aca cgt ttt gcg gat tgg gtg aac gca cta ggc gaa2448 Leu Ile Met Leu Thr Arg Phe Ala Asp Trp Val Asn Ala Leu Gly Glu 805810 815 aaa gcg tcc tcg gtg cta gcg gca ttt gaa gct aac tcg tta acg gca2496 Lys Ala Ser Ser Val Leu Ala Ala Phe Glu Ala Asn Ser Leu Thr Ala 820825 830 gaa caa ctg gct gat gcc atg aat ctt gat gct aat ttg ctg ttg caa2544 Glu Gln Leu Ala Asp Ala Met Asn Leu Asp Ala Asn Leu Leu Leu Gln 835840 845 gcc agt att caa gca caa aat cat caa cat ctt ccc cca gta act cca2592 Ala Ser Ile Gln Ala Gln Asn His Gln His Leu Pro Pro Val Thr Pro 850855 860 gaa aat gcg ttc tcc tgt tgg aca tct atc aat act atc ctg caa tgg2640 Glu Asn Ala Phe Ser Cys Trp Thr Ser Ile Asn Thr Ile Leu Gln Trp 865870 875 880 gtt aat gtc gca caa caa ttg aat gtc gcc cca cag ggc gtt tccgct 2688 Val Asn Val Ala Gln Gln Leu Asn Val Ala Pro Gln Gly Val Ser Ala885 890 895 ttg gtc ggg ctg gat tat att caa tca atg aaa gag aca ccg acctat 2736 Leu Val Gly Leu Asp Tyr Ile Gln Ser Met Lys Glu Thr Pro Thr Tyr900 905 910 gcc cag tgg gaa aac gcg gca ggc gta tta acc gcc ggg ttg aattca 2784 Ala Gln Trp Glu Asn Ala Ala Gly Val Leu Thr Ala Gly Leu Asn Ser915 920 925 caa cag gct aat aca tta cac gct ttt ctg gat gaa tct cgc agtgcc 2832 Gln Gln Ala Asn Thr Leu His Ala Phe Leu Asp Glu Ser Arg Ser Ala930 935 940 gca tta agc acc tac tat atc cgt caa gtc gcc aag gca gcg gcggct 2880 Ala Leu Ser Thr Tyr Tyr Ile Arg Gln Val Ala Lys Ala Ala Ala Ala945 950 955 960 att aaa agc cgt gat gac ttg tat caa tac tta ctg att gataat cag 2928 Ile Lys Ser Arg Asp Asp Leu Tyr Gln Tyr Leu Leu Ile Asp AsnGln 965 970 975 gtt tct gcg gca ata aaa acc acc cgg atc gcc gaa gcc attgcc agt 2976 Val Ser Ala Ala Ile Lys Thr Thr Arg Ile Ala Glu Ala Ile AlaSer 980 985 990 att caa ctg tac gtc aac cgg gca ttg gaa aat gtg gaa gaaaat gcc 3024 Ile Gln Leu Tyr Val Asn Arg Ala Leu Glu Asn Val Glu Glu AsnAla 995 1000 1005 aat tcg ggg gtt atc agc cgc caa ttc ttt atc gac tgggac aaa tac 3072 Asn Ser Gly Val Ile Ser Arg Gln Phe Phe Ile Asp Trp AspLys Tyr 1010 1015 1020 aat aaa cgc tac agc act tgg gcg ggt gtt tct caatta gtt tac tac 3120 Asn Lys Arg Tyr Ser Thr Trp Ala Gly Val Ser Gln LeuVal Tyr Tyr 1025 1030 1035 1040 ccg gaa aac tat att gat ccg acc atg cgtatc gga caa acc aaa atg 3168 Pro Glu Asn Tyr Ile Asp Pro Thr Met Arg IleGly Gln Thr Lys Met 1045 1050 1055 atg gac gca tta ctg caa tcc gtc agccaa agc caa tta aac gcc gat 3216 Met Asp Ala Leu Leu Gln Ser Val Ser GlnSer Gln Leu Asn Ala Asp 1060 1065 1070 acc gtc gaa gat gcc ttt atg tcttat ctg aca tcg ttt gaa caa gtg 3264 Thr Val Glu Asp Ala Phe Met Ser TyrLeu Thr Ser Phe Glu Gln Val 1075 1080 1085 gct aat ctt aaa gtt att agcgca tat cac gat aat att aat aac gat 3312 Ala Asn Leu Lys Val Ile Ser AlaTyr His Asp Asn Ile Asn Asn Asp 1090 1095 1100 caa ggg ctg acc tat tttatc gga ctc agt gaa act gat gcc ggt gaa 3360 Gln Gly Leu Thr Tyr Phe IleGly Leu Ser Glu Thr Asp Ala Gly Glu 1105 1110 1115 1120 tat tat tgg cgcagt gtc gat cac agt aaa ttc aac gac ggt aaa ttc 3408 Tyr Tyr Trp Arg SerVal Asp His Ser Lys Phe Asn Asp Gly Lys Phe 1125 1130 1135 gcg gct aatgcc tgg agt gaa tgg cat aaa att gat tgt cca att aac 3456 Ala Ala Asn AlaTrp Ser Glu Trp His Lys Ile Asp Cys Pro Ile Asn 1140 1145 1150 cct tataaa agc act atc cgt cca gtg ata tat aaa tcc cgc ctg tat 3504 Pro Tyr LysSer Thr Ile Arg Pro Val Ile Tyr Lys Ser Arg Leu Tyr 1155 1160 1165 ctgctc tgg ttg gaa caa aag gag atc acc aaa cag aca gga aat agt 3552 Leu LeuTrp Leu Glu Gln Lys Glu Ile Thr Lys Gln Thr Gly Asn Ser 1170 1175 1180aaa gat ggc tat caa act gaa acg gat tat cgt tat gaa cta aaa ttg 3600 LysAsp Gly Tyr Gln Thr Glu Thr Asp Tyr Arg Tyr Glu Leu Lys Leu 1185 11901195 1200 gcg cat atc cgc tat gat ggc act tgg aat acg cca atc acc tttgat 3648 Ala His Ile Arg Tyr Asp Gly Thr Trp Asn Thr Pro Ile Thr Phe Asp1205 1210 1215 gtc aat aaa aaa ata tcc gag cta aaa ctg gaa aaa aat agagcg ccc 3696 Val Asn Lys Lys Ile Ser Glu Leu Lys Leu Glu Lys Asn Arg AlaPro 1220 1225 1230 gga ctc tat tgt gcc ggt tat caa ggt gaa gat acg ttgctg gtg atg 3744 Gly Leu Tyr Cys Ala Gly Tyr Gln Gly Glu Asp Thr Leu LeuVal Met 1235 1240 1245 ttt tat aac caa caa gac aca cta gat agt tat aaaaac gct tca atg 3792 Phe Tyr Asn Gln Gln Asp Thr Leu Asp Ser Tyr Lys AsnAla Ser Met 1250 1255 1260 caa gga cta tat atc ttt gct gat atg gca tccaaa gat atg acc cca 3840 Gln Gly Leu Tyr Ile Phe Ala Asp Met Ala Ser LysAsp Met Thr Pro 1265 1270 1275 1280 gaa cag agc aat gtt tat cgg gat aatagc tat caa caa ttt gat acc 3888 Glu Gln Ser Asn Val Tyr Arg Asp Asn SerTyr Gln Gln Phe Asp Thr 1285 1290 1295 aat aat gtc aga aga gtg aat aaccgc tat gca gag gat tat gag att 3936 Asn Asn Val Arg Arg Val Asn Asn ArgTyr Ala Glu Asp Tyr Glu Ile 1300 1305 1310 cct tcc tcg gta agt agc cgtaaa gac tat ggt tgg gga gat tat tac 3984 Pro Ser Ser Val Ser Ser Arg LysAsp Tyr Gly Trp Gly Asp Tyr Tyr 1315 1320 1325 ctc agc atg gta tat aacgga gat att cca act atc aat tac aaa gcc 4032 Leu Ser Met Val Tyr Asn GlyAsp Ile Pro Thr Ile Asn Tyr Lys Ala 1330 1335 1340 gca tca agt gat ttaaaa atc tat atc tca cca aaa tta aga att att 4080 Ala Ser Ser Asp Leu LysIle Tyr Ile Ser Pro Lys Leu Arg Ile Ile 1345 1350 1355 1360 cat aat ggatat gaa gga cag aag cgc aat caa tgc aat ctg atg aat 4128 His Asn Gly TyrGlu Gly Gln Lys Arg Asn Gln Cys Asn Leu Met Asn 1365 1370 1375 aaa tatggc aaa cta ggt gat aaa ttt att gtt tat act agc ttg ggg 4176 Lys Tyr GlyLys Leu Gly Asp Lys Phe Ile Val Tyr Thr Ser Leu Gly 1380 1385 1390 gtcaat cca aat aac tcg tca aat aag ctc atg ttt tac ccc gtc tat 4224 Val AsnPro Asn Asn Ser Ser Asn Lys Leu Met Phe Tyr Pro Val Tyr 1395 1400 1405caa tat agc gga aac acc agt gga ctc aat caa ggg aga cta cta ttc 4272 GlnTyr Ser Gly Asn Thr Ser Gly Leu Asn Gln Gly Arg Leu Leu Phe 1410 14151420 cac cgt gac acc act tat cca tct aaa gta gaa gct tgg att cct gga4320 His Arg Asp Thr Thr Tyr Pro Ser Lys Val Glu Ala Trp Ile Pro Gly1425 1430 1435 1440 gca aaa cgt tct cta acc aac caa aat gcc gcc att ggtgat gat tat 4368 Ala Lys Arg Ser Leu Thr Asn Gln Asn Ala Ala Ile Gly AspAsp Tyr 1445 1450 1455 gct aca gac tct ctg aat aaa ccg gat gat ctt aagcaa tat atc ttt 4416 Ala Thr Asp Ser Leu Asn Lys Pro Asp Asp Leu Lys GlnTyr Ile Phe 1460 1465 1470 atg act gac agt aaa ggg act gct act gat gtctca ggc cca gta gag 4464 Met Thr Asp Ser Lys Gly Thr Ala Thr Asp Val SerGly Pro Val Glu 1475 1480 1485 att aat act gca att tct cca gca aaa gttcag ata ata gtc aaa gcg 4512 Ile Asn Thr Ala Ile Ser Pro Ala Lys Val GlnIle Ile Val Lys Ala 1490 1495 1500 ggt ggc aag gag caa act ttt acc gcagat aaa gat gtc tcc att cag 4560 Gly Gly Lys Glu Gln Thr Phe Thr Ala AspLys Asp Val Ser Ile Gln 1505 1510 1515 1520 cca tca cct agc ttt gat gaaatg aat tat caa ttt aat gcc ctt gaa 4608 Pro Ser Pro Ser Phe Asp Glu MetAsn Tyr Gln Phe Asn Ala Leu Glu 1525 1530 1535 ata gac ggt tct ggt ctgaat ttt att aac aac tca gcc agt att gat 4656 Ile Asp Gly Ser Gly Leu AsnPhe Ile Asn Asn Ser Ala Ser Ile Asp 1540 1545 1550 gtt act ttt acc gcattt gcg gag gat ggc cgc aaa ctg ggt tat gaa 4704 Val Thr Phe Thr Ala PheAla Glu Asp Gly Arg Lys Leu Gly Tyr Glu 1555 1560 1565 agt ttc agt attcct gtt acc ctc aag gta agt acc gat aat gcc ctg 4752 Ser Phe Ser Ile ProVal Thr Leu Lys Val Ser Thr Asp Asn Ala Leu 1570 1575 1580 acc ctg caccat aat gaa aat ggt gcg caa tat atg caa tgg caa tcc 4800 Thr Leu His HisAsn Glu Asn Gly Ala Gln Tyr Met Gln Trp Gln Ser 1585 1590 1595 1600 tatcgt acc cgc ctg aat act cta ttt gcc cgc cag ttg gtt gca cgc 4848 Tyr ArgThr Arg Leu Asn Thr Leu Phe Ala Arg Gln Leu Val Ala Arg 1605 1610 1615gcc acc acc gga atc gat aca att ctg agt atg gaa act cag aat att 4896 AlaThr Thr Gly Ile Asp Thr Ile Leu Ser Met Glu Thr Gln Asn Ile 1620 16251630 cag gaa ccg cag tta ggc aaa ggt ttc tat gct acg ttc gtg ata cct4944 Gln Glu Pro Gln Leu Gly Lys Gly Phe Tyr Ala Thr Phe Val Ile Pro1635 1640 1645 ccc tat aac cta tca act cat ggt gat gaa cgt tgg ttt aagctt tat 4992 Pro Tyr Asn Leu Ser Thr His Gly Asp Glu Arg Trp Phe Lys LeuTyr 1650 1655 1660 atc aaa cat gtt gtt gat aat aat tca cat att atc tattca ggc cag 5040 Ile Lys His Val Val Asp Asn Asn Ser His Ile Ile Tyr SerGly Gln 1665 1670 1675 1680 cta aca gat aca aat ata aac atc aca tta tttatt cct ctt gat gat 5088 Leu Thr Asp Thr Asn Ile Asn Ile Thr Leu Phe IlePro Leu Asp Asp 1685 1690 1695 gtc cca ttg aat caa gat tat cac gcc aaggtt tat atg acc ttc aag 5136 Val Pro Leu Asn Gln Asp Tyr His Ala Lys ValTyr Met Thr Phe Lys 1700 1705 1710 aaa tca cca tca gat ggt acc tgg tggggc cct cac ttt gtt aga gat 5184 Lys Ser Pro Ser Asp Gly Thr Trp Trp GlyPro His Phe Val Arg Asp 1715 1720 1725 gat aaa gga ata gta aca ata aaccct aaa tcc att ttg acc cat ttt 5232 Asp Lys Gly Ile Val Thr Ile Asn ProLys Ser Ile Leu Thr His Phe 1730 1735 1740 gag agc gtc aat gtc ctg aataat att agt agc gaa cca atg gat ttc 5280 Glu Ser Val Asn Val Leu Asn AsnIle Ser Ser Glu Pro Met Asp Phe 1745 1750 1755 1760 agc ggc gct aac agcctc tat ttc tgg gaa ctg ttc tac tat acc ccg 5328 Ser Gly Ala Asn Ser LeuTyr Phe Trp Glu Leu Phe Tyr Tyr Thr Pro 1765 1770 1775 atg ctg gtt gctcaa cgt ttg ctg cat gaa cag aac ttc gat gaa gcc 5376 Met Leu Val Ala GlnArg Leu Leu His Glu Gln Asn Phe Asp Glu Ala 1780 1785 1790 aac cgt tggctg aaa tat gtc tgg agt cca tcc ggt tat att gtc cac 5424 Asn Arg Trp LeuLys Tyr Val Trp Ser Pro Ser Gly Tyr Ile Val His 1795 1800 1805 ggc cagatt cag aac tac cag tgg aac gtc cgc ccg tta ctg gaa gac 5472 Gly Gln IleGln Asn Tyr Gln Trp Asn Val Arg Pro Leu Leu Glu Asp 1810 1815 1820 accagt tgg aac agt gat cct ttg gat tcc gtc gat cct gac gcg gta 5520 Thr SerTrp Asn Ser Asp Pro Leu Asp Ser Val Asp Pro Asp Ala Val 1825 1830 18351840 gca cag cac gat cca atg cac tac aaa gtt tca act ttt atg cgt acc5568 Ala Gln His Asp Pro Met His Tyr Lys Val Ser Thr Phe Met Arg Thr1845 1850 1855 ttg gat cta ttg ata gca cgc ggc gac cat gct tat cgc caactg gaa 5616 Leu Asp Leu Leu Ile Ala Arg Gly Asp His Ala Tyr Arg Gln LeuGlu 1860 1865 1870 cga gat aca ctc aac gaa gcg aag atg tgg tat atg caagcg ctg cat 5664 Arg Asp Thr Leu Asn Glu Ala Lys Met Trp Tyr Met Gln AlaLeu His 1875 1880 1885 cta tta ggt gac aaa cct tat cta ccg ctg agt acgaca tgg agt gat 5712 Leu Leu Gly Asp Lys Pro Tyr Leu Pro Leu Ser Thr ThrTrp Ser Asp 1890 1895 1900 cca cga cta gac aga gcc gcg gat atc act acccaa aat gct cac gac 5760 Pro Arg Leu Asp Arg Ala Ala Asp Ile Thr Thr GlnAsn Ala His Asp 1905 1910 1915 1920 agc gca ata gtc gct ctg cgg cag aatata cct aca ccg gca cct tta 5808 Ser Ala Ile Val Ala Leu Arg Gln Asn IlePro Thr Pro Ala Pro Leu 1925 1930 1935 tca ttg cgc agc gct aat acc ctgact gat ctc ttc ctg ccg caa atc 5856 Ser Leu Arg Ser Ala Asn Thr Leu ThrAsp Leu Phe Leu Pro Gln Ile 1940 1945 1950 aat gaa gtg atg atg aat tactgg cag aca tta gct cag aga gta tac 5904 Asn Glu Val Met Met Asn Tyr TrpGln Thr Leu Ala Gln Arg Val Tyr 1955 1960 1965 aat ctg cgt cat aac ctctct atc gac ggc cag ccg tta tat ctg cca 5952 Asn Leu Arg His Asn Leu SerIle Asp Gly Gln Pro Leu Tyr Leu Pro 1970 1975 1980 atc tat gcc aca ccggcc gat ccg aaa gcg tta ctc agc gcc gcc gtt 6000 Ile Tyr Ala Thr Pro AlaAsp Pro Lys Ala Leu Leu Ser Ala Ala Val 1985 1990 1995 2000 gcc act tctcaa ggt gga ggc aag cta ccg gaa tca ttt atg tcc ctg 6048 Ala Thr Ser GlnGly Gly Gly Lys Leu Pro Glu Ser Phe Met Ser Leu 2005 2010 2015 tgg cgtttc ccg cac atg ctg gaa aat gcg cgc ggc atg gtt agc cag 6096 Trp Arg PhePro His Met Leu Glu Asn Ala Arg Gly Met Val Ser Gln 2020 2025 2030 ctcacc cag ttc ggc tcc acg tta caa aat att atc gaa cgt cag gac 6144 Leu ThrGln Phe Gly Ser Thr Leu Gln Asn Ile Ile Glu Arg Gln Asp 2035 2040 2045gcg gaa gcg ctc aat gcg tta tta caa aat cag gcc gcc gag ctg ata 6192 AlaGlu Ala Leu Asn Ala Leu Leu Gln Asn Gln Ala Ala Glu Leu Ile 2050 20552060 ttg act aac ctg agc att cag gac aaa acc att gaa gaa ttg gat gcc6240 Leu Thr Asn Leu Ser Ile Gln Asp Lys Thr Ile Glu Glu Leu Asp Ala2065 2070 2075 2080 gag aaa acg gtg ttg gaa aaa tcc aaa gcg gga gca caatcg cgc ttt 6288 Glu Lys Thr Val Leu Glu Lys Ser Lys Ala Gly Ala Gln SerArg Phe 2085 2090 2095 gat agc tac ggc aaa ctg tac gat gag aat atc aacgcc ggt gaa aac 6336 Asp Ser Tyr Gly Lys Leu Tyr Asp Glu Asn Ile Asn AlaGly Glu Asn 2100 2105 2110 caa gcc atg acg cta cga gcg tcc gcc gcc gggctt acc acg gca gtt 6384 Gln Ala Met Thr Leu Arg Ala Ser Ala Ala Gly LeuThr Thr Ala Val 2115 2120 2125 cag gca tcc cgt ctg gcc ggt gcg gcg gctgat ctg gtg cct aac atc 6432 Gln Ala Ser Arg Leu Ala Gly Ala Ala Ala AspLeu Val Pro Asn Ile 2130 2135 2140 ttc ggc ttt gcc ggt ggc ggc agc cgttgg ggg gct atc gct gag gcg 6480 Phe Gly Phe Ala Gly Gly Gly Ser Arg TrpGly Ala Ile Ala Glu Ala 2145 2150 2155 2160 aca ggt tat gtg atg gaa ttctcc gcg aat gtt atg aac acc gaa gcg 6528 Thr Gly Tyr Val Met Glu Phe SerAla Asn Val Met Asn Thr Glu Ala 2165 2170 2175 gat aaa att agc caa tctgaa acc tac cgt cgt cgc cgt cag gag tgg 6576 Asp Lys Ile Ser Gln Ser GluThr Tyr Arg Arg Arg Arg Gln Glu Trp 2180 2185 2190 gag atc cag cgg aataat gcc gaa gcg gaa ttg aag caa atc gat gct 6624 Glu Ile Gln Arg Asn AsnAla Glu Ala Glu Leu Lys Gln Ile Asp Ala 2195 2200 2205 cag ctc aaa tcactc gct gta cgc cgc gaa gcc gcc gta ttg cag aaa 6672 Gln Leu Lys Ser LeuAla Val Arg Arg Glu Ala Ala Val Leu Gln Lys 2210 2215 2220 acc agt ctgaaa acc caa caa gaa cag acc caa tct caa ttg gcc ttc 6720 Thr Ser Leu LysThr Gln Gln Glu Gln Thr Gln Ser Gln Leu Ala Phe 2225 2230 2235 2240 ctgcaa cgt aag ttc agc aat cag gcg tta tac aac tgg ctg cgt ggt 6768 Leu GlnArg Lys Phe Ser Asn Gln Ala Leu Tyr Asn Trp Leu Arg Gly 2245 2250 2255cga ctg gcg gcg att tac ttc cag ttc tac gat ttg gcc gtc gcg cgt 6816 ArgLeu Ala Ala Ile Tyr Phe Gln Phe Tyr Asp Leu Ala Val Ala Arg 2260 22652270 tgc ctg atg gca gaa caa gct tac cgt tgg gaa ctc aat gat gac tct6864 Cys Leu Met Ala Glu Gln Ala Tyr Arg Trp Glu Leu Asn Asp Asp Ser2275 2280 2285 gcc cgc ttc att aaa ccg ggc gcc tgg cag gga acc tat gccggt ctg 6912 Ala Arg Phe Ile Lys Pro Gly Ala Trp Gln Gly Thr Tyr Ala GlyLeu 2290 2295 2300 ctt gca ggt gaa acc ttg atg ctg agt ctg gca caa atggaa gac gct 6960 Leu Ala Gly Glu Thr Leu Met Leu Ser Leu Ala Gln Met GluAsp Ala 2305 2310 2315 2320 cat ctg aaa cgc gat aaa cgc gca tta gag gttgaa cgc aca gta tcg 7008 His Leu Lys Arg Asp Lys Arg Ala Leu Glu Val GluArg Thr Val Ser 2325 2330 2335 ctg gcc gaa gtt tat gca gga tta cca aaagat aac ggt cca ttt tcc 7056 Leu Ala Glu Val Tyr Ala Gly Leu Pro Lys AspAsn Gly Pro Phe Ser 2340 2345 2350 ctg gct cag gaa att gac aag ctg gtgagt caa ggt tca ggc agt gcc 7104 Leu Ala Gln Glu Ile Asp Lys Leu Val SerGln Gly Ser Gly Ser Ala 2355 2360 2365 ggc agt ggt aat aat aat ttg gcgttc ggc gcc ggc acg gac act aaa 7152 Gly Ser Gly Asn Asn Asn Leu Ala PheGly Ala Gly Thr Asp Thr Lys 2370 2375 2380 acc tct ttg cag gca tca gtttca ttc gct gat ttg aaa att cgt gaa 7200 Thr Ser Leu Gln Ala Ser Val SerPhe Ala Asp Leu Lys Ile Arg Glu 2385 2390 2395 2400 gat tac ccg gca tcgctt ggc aaa att cga cgt atc aaa cag atc agc 7248 Asp Tyr Pro Ala Ser LeuGly Lys Ile Arg Arg Ile Lys Gln Ile Ser 2405 2410 2415 gtc act ttg cccgcg cta ctg gga ccg tat cag gat gta cag gca ata 7296 Val Thr Leu Pro AlaLeu Leu Gly Pro Tyr Gln Asp Val Gln Ala Ile 2420 2425 2430 ttg tct tacggc gat aaa gcc gga tta gct aac ggc tgt gaa gcg ctg 7344 Leu Ser Tyr GlyAsp Lys Ala Gly Leu Ala Asn Gly Cys Glu Ala Leu 2435 2440 2445 gca gtttct cac ggt atg aat gac agc ggc caa ttc cag ctc gat ttc 7392 Ala Val SerHis Gly Met Asn Asp Ser Gly Gln Phe Gln Leu Asp Phe 2450 2455 2460 aacgat ggc aaa ttc ctg cca ttc gaa ggc atc gcc att gat caa ggc 7440 Asn AspGly Lys Phe Leu Pro Phe Glu Gly Ile Ala Ile Asp Gln Gly 2465 2470 24752480 acg ctg aca ctg agc ttc cca aat gca tct atg ccg gag aaa ggt aaa7488 Thr Leu Thr Leu Ser Phe Pro Asn Ala Ser Met Pro Glu Lys Gly Lys2485 2490 2495 caa gcc act atg tta aaa acc ctg aac gat atc att ttg catatt cgc 7536 Gln Ala Thr Met Leu Lys Thr Leu Asn Asp Ile Ile Leu His IleArg 2500 2505 2510 tac acc att aaa taa 7551 Tyr Thr Ile Lys 2515 2 7515DNA Photorhabdus luminescens CDS (1)..(7512) 2 atg caa aac tca tta tcaagc act atc gat act att tgt cag aaa ctg 48 Met Gln Asn Ser Leu Ser SerThr Ile Asp Thr Ile Cys Gln Lys Leu 1 5 10 15 caa tta act tgt ccg gcggaa att gct ttg tat ccc ttt gat act ttc 96 Gln Leu Thr Cys Pro Ala GluIle Ala Leu Tyr Pro Phe Asp Thr Phe 20 25 30 cgg gaa aaa act cgg gga atggtt aat tgg ggg gaa gca aaa cgg att 144 Arg Glu Lys Thr Arg Gly Met ValAsn Trp Gly Glu Ala Lys Arg Ile 35 40 45 tat gaa att gca caa gcg gaa caggat aga aac cta ctt cat gaa aaa 192 Tyr Glu Ile Ala Gln Ala Glu Gln AspArg Asn Leu Leu His Glu Lys 50 55 60 cgt att ttt gcc tat gct aat ccg ctgctg aaa aac gct gtt cgg ttg 240 Arg Ile Phe Ala Tyr Ala Asn Pro Leu LeuLys Asn Ala Val Arg Leu 65 70 75 80 ggt acc cgg caa atg ttg ggt ttt atacaa ggt tat agt gat ctg ttt 288 Gly Thr Arg Gln Met Leu Gly Phe Ile GlnGly Tyr Ser Asp Leu Phe 85 90 95 ggt aat cgt gct gat aac tat gcc gcg ccgggc tcg gtt gca tcg atg 336 Gly Asn Arg Ala Asp Asn Tyr Ala Ala Pro GlySer Val Ala Ser Met 100 105 110 ttc tca ccg gcg gct tat ttg acg gaa ttgtac cgt gaa gcc aaa aac 384 Phe Ser Pro Ala Ala Tyr Leu Thr Glu Leu TyrArg Glu Ala Lys Asn 115 120 125 ttg cat gac agc agc tca att tat tac ctagat aaa cgt cgc ccg gat 432 Leu His Asp Ser Ser Ser Ile Tyr Tyr Leu AspLys Arg Arg Pro Asp 130 135 140 tta gca agc tta atg ctc agc cag aaa aatatg gat gag gaa att tca 480 Leu Ala Ser Leu Met Leu Ser Gln Lys Asn MetAsp Glu Glu Ile Ser 145 150 155 160 acg ctg gct ctc tct aat gaa ttg tgcctt gcc ggg atc gaa aca aaa 528 Thr Leu Ala Leu Ser Asn Glu Leu Cys LeuAla Gly Ile Glu Thr Lys 165 170 175 aca gga aaa tca caa gat gaa gtg atggat atg ttg tca act tat cgt 576 Thr Gly Lys Ser Gln Asp Glu Val Met AspMet Leu Ser Thr Tyr Arg 180 185 190 tta agt gga gag aca cct tat cat cacgct tat gaa act gtt cgt gaa 624 Leu Ser Gly Glu Thr Pro Tyr His His AlaTyr Glu Thr Val Arg Glu 195 200 205 atc gtt cat gaa cgt gat cca gga tttcgt cat ttg tca cag gca ccc 672 Ile Val His Glu Arg Asp Pro Gly Phe ArgHis Leu Ser Gln Ala Pro 210 215 220 att gtt gct gct aag ctc gat cct gtgact ttg ttg ggt att agc tcc 720 Ile Val Ala Ala Lys Leu Asp Pro Val ThrLeu Leu Gly Ile Ser Ser 225 230 235 240 cat att tcg cca gaa ctg tat aacttg ctg att gag gag atc ccg gaa 768 His Ile Ser Pro Glu Leu Tyr Asn LeuLeu Ile Glu Glu Ile Pro Glu 245 250 255 aaa gat gaa gcc gcg ctt gat acgctt tat aaa aca aac ttt ggc gat 816 Lys Asp Glu Ala Ala Leu Asp Thr LeuTyr Lys Thr Asn Phe Gly Asp 260 265 270 att act act gct cag tta atg tcccca agt tat ctg gcc cgg tat tat 864 Ile Thr Thr Ala Gln Leu Met Ser ProSer Tyr Leu Ala Arg Tyr Tyr 275 280 285 ggc gtc tca ccg gaa gat att gcctac gtg acg act tca tta tca cat 912 Gly Val Ser Pro Glu Asp Ile Ala TyrVal Thr Thr Ser Leu Ser His 290 295 300 gtt gga tat agc agt gat att ctggtt att ccg ttg gtc gat ggt gtg 960 Val Gly Tyr Ser Ser Asp Ile Leu ValIle Pro Leu Val Asp Gly Val 305 310 315 320 ggt aag atg gaa gta gtt cgtgtt acc cga aca cca tcg gat aat tat 1008 Gly Lys Met Glu Val Val Arg ValThr Arg Thr Pro Ser Asp Asn Tyr 325 330 335 acc agt cag acg aat tat attgag ctg tat cca cag ggt ggc gac aat 1056 Thr Ser Gln Thr Asn Tyr Ile GluLeu Tyr Pro Gln Gly Gly Asp Asn 340 345 350 tat ttg atc aaa tac aat ctaagc aat agt ttt ggt ttg gat gat ttt 1104 Tyr Leu Ile Lys Tyr Asn Leu SerAsn Ser Phe Gly Leu Asp Asp Phe 355 360 365 tat ctg caa tat aaa gat ggttcc gct gat tgg act gag att gcc cat 1152 Tyr Leu Gln Tyr Lys Asp Gly SerAla Asp Trp Thr Glu Ile Ala His 370 375 380 aat ccc tat cct gat atg gtcata aat caa aag tat gaa tca cag gcg 1200 Asn Pro Tyr Pro Asp Met Val IleAsn Gln Lys Tyr Glu Ser Gln Ala 385 390 395 400 aca atc aaa cgt agt gactct gac aat ata ctc agt ata ggg tta caa 1248 Thr Ile Lys Arg Ser Asp SerAsp Asn Ile Leu Ser Ile Gly Leu Gln 405 410 415 aga tgg cat agc ggt agttat aat ttt gcc gcc gcc aat ttt aaa att 1296 Arg Trp His Ser Gly Ser TyrAsn Phe Ala Ala Ala Asn Phe Lys Ile 420 425 430 gac caa tac tcc ccg aaagct ttc ctg ctt aaa atg aat aag gct att 1344 Asp Gln Tyr Ser Pro Lys AlaPhe Leu Leu Lys Met Asn Lys Ala Ile 435 440 445 cgg ttg ctc aaa gct accggc ctc tct ttt gct acg ttg gag cgt att 1392 Arg Leu Leu Lys Ala Thr GlyLeu Ser Phe Ala Thr Leu Glu Arg Ile 450 455 460 gtt gat agt gtt aat agcacc aaa tcc atc acg gtt gag gta tta aac 1440 Val Asp Ser Val Asn Ser ThrLys Ser Ile Thr Val Glu Val Leu Asn 465 470 475 480 aag gtt tat cgg gtaaaa ttc tat att gat cgt tat ggc atc agt gaa 1488 Lys Val Tyr Arg Val LysPhe Tyr Ile Asp Arg Tyr Gly Ile Ser Glu 485 490 495 gag aca gcc gct attttg gct aat att aat atc tct cag caa gct gtt 1536 Glu Thr Ala Ala Ile LeuAla Asn Ile Asn Ile Ser Gln Gln Ala Val 500 505 510 ggc aat cag ctt agccag ttt gag caa cta ttt aat cac ccg ccg ctc 1584 Gly Asn Gln Leu Ser GlnPhe Glu Gln Leu Phe Asn His Pro Pro Leu 515 520 525 aat ggt att cgc tatgaa atc agt gag gac aac tcc aaa cat ctt cct 1632 Asn Gly Ile Arg Tyr GluIle Ser Glu Asp Asn Ser Lys His Leu Pro 530 535 540 aat cct gat ctg aacctt aaa cca gac agt acc ggt gat gat caa cgc 1680 Asn Pro Asp Leu Asn LeuLys Pro Asp Ser Thr Gly Asp Asp Gln Arg 545 550 555 560 aag gcg gtt ttaaaa cgc gcg ttt cag gtt aac gcc agt gag ttg tat 1728 Lys Ala Val Leu LysArg Ala Phe Gln Val Asn Ala Ser Glu Leu Tyr 565 570 575 cag atg tta ttgatc act gat cgt aaa gaa gac ggt gtt atc aaa aat 1776 Gln Met Leu Leu IleThr Asp Arg Lys Glu Asp Gly Val Ile Lys Asn 580 585 590 aac tta gag aatttg tct gat ctg tat ttg gtt agt ttg ctg gcc cag 1824 Asn Leu Glu Asn LeuSer Asp Leu Tyr Leu Val Ser Leu Leu Ala Gln 595 600 605 att cat aac ctgact att gct gaa ttg aac att ttg ttg gtg att tgt 1872 Ile His Asn Leu ThrIle Ala Glu Leu Asn Ile Leu Leu Val Ile Cys 610 615 620 ggc tat ggc gacacc aac att tat cag att acc gac gat aat tta gcc 1920 Gly Tyr Gly Asp ThrAsn Ile Tyr Gln Ile Thr Asp Asp Asn Leu Ala 625 630 635 640 aaa ata gtggaa aca ttg ttg tgg atc act caa tgg ttg aag acc caa 1968 Lys Ile Val GluThr Leu Leu Trp Ile Thr Gln Trp Leu Lys Thr Gln 645 650 655 aaa tgg acagtt acc gac ctg ttt ctg atg acc acg gcc act tac agc 2016 Lys Trp Thr ValThr Asp Leu Phe Leu Met Thr Thr Ala Thr Tyr Ser 660 665 670 acc act ttaacg cca gaa att agc aat ctg acg gct acg ttg tct tca 2064 Thr Thr Leu ThrPro Glu Ile Ser Asn Leu Thr Ala Thr Leu Ser Ser 675 680 685 act ttg catggc aaa gag agt ctg att ggg gaa gat ctg aaa aga gca 2112 Thr Leu His GlyLys Glu Ser Leu Ile Gly Glu Asp Leu Lys Arg Ala 690 695 700 atg gcg ccttgc ttc act tcg gct ttg cat ttg act tct caa gaa gtt 2160 Met Ala Pro CysPhe Thr Ser Ala Leu His Leu Thr Ser Gln Glu Val 705 710 715 720 gcg tatgac ctg ctg ttg tgg ata gac cag att caa ccg gca caa ata 2208 Ala Tyr AspLeu Leu Leu Trp Ile Asp Gln Ile Gln Pro Ala Gln Ile 725 730 735 act gttgat ggg ttt tgg gaa gaa gtg caa aca aca cca acc agc ttg 2256 Thr Val AspGly Phe Trp Glu Glu Val Gln Thr Thr Pro Thr Ser Leu 740 745 750 aag gtgatt acc ttt gct cag gtg ctg gca caa ttg agc ctg atc tat 2304 Lys Val IleThr Phe Ala Gln Val Leu Ala Gln Leu Ser Leu Ile Tyr 755 760 765 cgt cgtatt ggg tta agt gaa acg gaa ctg tca ctg atc gtg act caa 2352 Arg Arg IleGly Leu Ser Glu Thr Glu Leu Ser Leu Ile Val Thr Gln 770 775 780 tct tctctg cta gtg gca ggc aaa agc ata ctg gat cac ggt ctg tta 2400 Ser Ser LeuLeu Val Ala Gly Lys Ser Ile Leu Asp His Gly Leu Leu 785 790 795 800 accctg atg gcc ttg gaa ggt ttt cat acc tgg gtt aat ggc ttg ggg 2448 Thr LeuMet Ala Leu Glu Gly Phe His Thr Trp Val Asn Gly Leu Gly 805 810 815 caacat gcc tcc ttg ata ttg gcg gcg ttg aaa gac gga gcc ttg aca 2496 Gln HisAla Ser Leu Ile Leu Ala Ala Leu Lys Asp Gly Ala Leu Thr 820 825 830 gttacc gat gta gca caa gct atg aat aag gag gaa tct ctc cta caa 2544 Val ThrAsp Val Ala Gln Ala Met Asn Lys Glu Glu Ser Leu Leu Gln 835 840 845 atggca gct aat cag gtg gag aag gat cta aca aaa ctg acc agt tgg 2592 Met AlaAla Asn Gln Val Glu Lys Asp Leu Thr Lys Leu Thr Ser Trp 850 855 860 acacag att gac gct att ctg caa tgg tta cag atg tct tcg gcc ttg 2640 Thr GlnIle Asp Ala Ile Leu Gln Trp Leu Gln Met Ser Ser Ala Leu 865 870 875 880gcg gtt tct cca ctg gat ctg gca ggg atg atg gcc ctg aaa tat ggg 2688 AlaVal Ser Pro Leu Asp Leu Ala Gly Met Met Ala Leu Lys Tyr Gly 885 890 895ata gat cat aac tat gct gcc tgg caa gct gcg gcg gct gcg ctg atg 2736 IleAsp His Asn Tyr Ala Ala Trp Gln Ala Ala Ala Ala Ala Leu Met 900 905 910gct gat cat gct aat cag gca cag aaa aaa ctg gat gag acg ttc agt 2784 AlaAsp His Ala Asn Gln Ala Gln Lys Lys Leu Asp Glu Thr Phe Ser 915 920 925aag gca tta tgt aac tat tat att aat gct gtt gtc gat agt gct gct 2832 LysAla Leu Cys Asn Tyr Tyr Ile Asn Ala Val Val Asp Ser Ala Ala 930 935 940gga gta cgt gat cgt aac ggt tta tat acc tat ttg ctg att gat aat 2880 GlyVal Arg Asp Arg Asn Gly Leu Tyr Thr Tyr Leu Leu Ile Asp Asn 945 950 955960 cag gtt tct gcc gat gtg atc act tca cgt att gca gaa gct atc gcc 2928Gln Val Ser Ala Asp Val Ile Thr Ser Arg Ile Ala Glu Ala Ile Ala 965 970975 ggt att caa ctg tac gtt aac cgg gct tta aac cga gat gaa ggt cag 2976Gly Ile Gln Leu Tyr Val Asn Arg Ala Leu Asn Arg Asp Glu Gly Gln 980 985990 ctt gca tcg gac gtt agt acc cgt cag ttc ttc act gac tgg gaa cgt 3024Leu Ala Ser Asp Val Ser Thr Arg Gln Phe Phe Thr Asp Trp Glu Arg 995 10001005 tac aat aaa cgt tac agt act tgg gct ggt gtc tct gaa ctg gtc tat3072 Tyr Asn Lys Arg Tyr Ser Thr Trp Ala Gly Val Ser Glu Leu Val Tyr1010 1015 1020 tat cca gaa aac tat gtt gat ccc act cag cgc att ggg caaacc aaa 3120 Tyr Pro Glu Asn Tyr Val Asp Pro Thr Gln Arg Ile Gly Gln ThrLys 1025 1030 1035 1040 atg atg gat gcg ctg ttg caa tcc atc aac cag agccag cta aat gcg 3168 Met Met Asp Ala Leu Leu Gln Ser Ile Asn Gln Ser GlnLeu Asn Ala 1045 1050 1055 gat acg gtg gaa gat gct ttc aaa act tat ttgacc agc ttt gag cag 3216 Asp Thr Val Glu Asp Ala Phe Lys Thr Tyr Leu ThrSer Phe Glu Gln 1060 1065 1070 gta gca aat ctg aaa gta att agt gct taccac gat aat gtg aat gtg 3264 Val Ala Asn Leu Lys Val Ile Ser Ala Tyr HisAsp Asn Val Asn Val 1075 1080 1085 gat caa gga tta act tat ttt atc ggtatc gac caa gca gct ccg ggt 3312 Asp Gln Gly Leu Thr Tyr Phe Ile Gly IleAsp Gln Ala Ala Pro Gly 1090 1095 1100 acg tat tac tgg cgt agt gtt gatcac agc aaa tgt gaa aat ggc aag 3360 Thr Tyr Tyr Trp Arg Ser Val Asp HisSer Lys Cys Glu Asn Gly Lys 1105 1110 1115 1120 ttt gcc gct aat gct tggggt gag tgg aat aaa att acc tgt gct gtc 3408 Phe Ala Ala Asn Ala Trp GlyGlu Trp Asn Lys Ile Thr Cys Ala Val 1125 1130 1135 aat cct tgg aaa aatatc atc cgt ccg gtt gtt tat atg tcc cgc tta 3456 Asn Pro Trp Lys Asn IleIle Arg Pro Val Val Tyr Met Ser Arg Leu 1140 1145 1150 tat ctg cta tggctg gag cag caa tca aag aaa agt gat gat ggt aaa 3504 Tyr Leu Leu Trp LeuGlu Gln Gln Ser Lys Lys Ser Asp Asp Gly Lys 1155 1160 1165 acc acg atttat caa tat aac tta aaa ctg gct cat att cgt tac gac 3552 Thr Thr Ile TyrGln Tyr Asn Leu Lys Leu Ala His Ile Arg Tyr Asp 1170 1175 1180 ggt agttgg aat aca cca ttt act ttt gat gtg aca gaa aag gta aaa 3600 Gly Ser TrpAsn Thr Pro Phe Thr Phe Asp Val Thr Glu Lys Val Lys 1185 1190 1195 1200aat tac acg tcg agt act gat gct gct gaa tct tta ggg ttg tat tgt 3648 AsnTyr Thr Ser Ser Thr Asp Ala Ala Glu Ser Leu Gly Leu Tyr Cys 1205 12101215 act ggt tat caa ggg gaa gac act cta tta gtt atg ttc tat tcg atg3696 Thr Gly Tyr Gln Gly Glu Asp Thr Leu Leu Val Met Phe Tyr Ser Met1220 1225 1230 cag agt agt tat agc tcc tat acc gat aat aat gcg ccg gtcact ggg 3744 Gln Ser Ser Tyr Ser Ser Tyr Thr Asp Asn Asn Ala Pro Val ThrGly 1235 1240 1245 cta tat att ttc gct gat atg tca tca gac aat atg acgaat gca caa 3792 Leu Tyr Ile Phe Ala Asp Met Ser Ser Asp Asn Met Thr AsnAla Gln 1250 1255 1260 gca act aac tat tgg aat aac agt tat ccg caa tttgat act gtg atg 3840 Ala Thr Asn Tyr Trp Asn Asn Ser Tyr Pro Gln Phe AspThr Val Met 1265 1270 1275 1280 gca gat ccg gat agc gac aat aaa aaa gtcata acc aga aga gtt aat 3888 Ala Asp Pro Asp Ser Asp Asn Lys Lys Val IleThr Arg Arg Val Asn 1285 1290 1295 aac cgt tat gcg gag gat tat gaa attcct tcc tct gtg aca agt aac 3936 Asn Arg Tyr Ala Glu Asp Tyr Glu Ile ProSer Ser Val Thr Ser Asn 1300 1305 1310 agt aat tat tct tgg ggt gat cacagt tta acc atg ctt tat ggt ggt 3984 Ser Asn Tyr Ser Trp Gly Asp His SerLeu Thr Met Leu Tyr Gly Gly 1315 1320 1325 agt gtt cct aat att act tttgaa tcg gcg gca gaa gat tta agg cta 4032 Ser Val Pro Asn Ile Thr Phe GluSer Ala Ala Glu Asp Leu Arg Leu 1330 1335 1340 tct acc aat atg gca ttgagt att att cat aat gga tat gcg gga acc 4080 Ser Thr Asn Met Ala Leu SerIle Ile His Asn Gly Tyr Ala Gly Thr 1345 1350 1355 1360 cgc cgt ata caatgt aat ctt atg aaa caa tac gct tca tta ggt gat 4128 Arg Arg Ile Gln CysAsn Leu Met Lys Gln Tyr Ala Ser Leu Gly Asp 1365 1370 1375 aaa ttt ataatt tat gat tca tca ttt gat gat gca aac cgt ttt aat 4176 Lys Phe Ile IleTyr Asp Ser Ser Phe Asp Asp Ala Asn Arg Phe Asn 1380 1385 1390 ctg gtgcca ttg ttt aaa ttc gga aaa gac gag aac tca gat gat agt 4224 Leu Val ProLeu Phe Lys Phe Gly Lys Asp Glu Asn Ser Asp Asp Ser 1395 1400 1405 atttgt ata tat aat gaa aac cct tcc tct gaa gat aag aag tgg tat 4272 Ile CysIle Tyr Asn Glu Asn Pro Ser Ser Glu Asp Lys Lys Trp Tyr 1410 1415 1420ttt tct tcg aaa gat gac aat aaa aca gcg gat tat aat ggt gga act 4320 PheSer Ser Lys Asp Asp Asn Lys Thr Ala Asp Tyr Asn Gly Gly Thr 1425 14301435 1440 caa tgt ata gat gct gga acc agt aac aaa gat ttt tat tat aatctc 4368 Gln Cys Ile Asp Ala Gly Thr Ser Asn Lys Asp Phe Tyr Tyr Asn Leu1445 1450 1455 cag gag att gaa gta att agt gtt act ggt ggg tat tgg tcgagt tat 4416 Gln Glu Ile Glu Val Ile Ser Val Thr Gly Gly Tyr Trp Ser SerTyr 1460 1465 1470 aaa ata tcc aac ccg att aat atc aat acg ggc att gatagt gct aaa 4464 Lys Ile Ser Asn Pro Ile Asn Ile Asn Thr Gly Ile Asp SerAla Lys 1475 1480 1485 gta aaa gtc acc gta aaa gcg ggt ggt gac gat caaatc ttt act gct 4512 Val Lys Val Thr Val Lys Ala Gly Gly Asp Asp Gln IlePhe Thr Ala 1490 1495 1500 gat aat agt acc tat gtt cct cag caa ccg gcaccc agt ttt gag gag 4560 Asp Asn Ser Thr Tyr Val Pro Gln Gln Pro Ala ProSer Phe Glu Glu 1505 1510 1515 1520 atg att tat cag ttc aat aac ctg acaata gat tgt aag aat tta aat 4608 Met Ile Tyr Gln Phe Asn Asn Leu Thr IleAsp Cys Lys Asn Leu Asn 1525 1530 1535 ttc atc gac aat cag gca cat attgag att gat ttc acc gct acg gca 4656 Phe Ile Asp Asn Gln Ala His Ile GluIle Asp Phe Thr Ala Thr Ala 1540 1545 1550 caa gat ggc cga ttc ttg ggtgca gaa act ttt att atc ccg gta act 4704 Gln Asp Gly Arg Phe Leu Gly AlaGlu Thr Phe Ile Ile Pro Val Thr 1555 1560 1565 aaa aaa gtt ctc ggt actgag aac gtg att gcg tta tat agc gaa aat 4752 Lys Lys Val Leu Gly Thr GluAsn Val Ile Ala Leu Tyr Ser Glu Asn 1570 1575 1580 aac ggt gtt caa tatatg caa att ggc gca tat cgt acc cgt ttg aat 4800 Asn Gly Val Gln Tyr MetGln Ile Gly Ala Tyr Arg Thr Arg Leu Asn 1585 1590 1595 1600 acg tta ttcgct caa cag ttg gtt agc cgt gct aat cgt ggc att gat 4848 Thr Leu Phe AlaGln Gln Leu Val Ser Arg Ala Asn Arg Gly Ile Asp 1605 1610 1615 gca gtgctc agt atg gaa act cag aat att cag gaa ccg caa tta gga 4896 Ala Val LeuSer Met Glu Thr Gln Asn Ile Gln Glu Pro Gln Leu Gly 1620 1625 1630 gcgggc aca tat gtg cag ctt gtg ttg gat aaa tat gat gag tct att 4944 Ala GlyThr Tyr Val Gln Leu Val Leu Asp Lys Tyr Asp Glu Ser Ile 1635 1640 1645cat ggc act aat aaa agc ttt gct att gaa tat gtt gat ata ttt aaa 4992 HisGly Thr Asn Lys Ser Phe Ala Ile Glu Tyr Val Asp Ile Phe Lys 1650 16551660 gag aac gat agt ttt gtg att tat caa gga gaa ctt agc gaa aca agt5040 Glu Asn Asp Ser Phe Val Ile Tyr Gln Gly Glu Leu Ser Glu Thr Ser1665 1670 1675 1680 caa act gtt gtg aaa gtt ttc tta tcc tat ttt ata gaggcg act gga 5088 Gln Thr Val Val Lys Val Phe Leu Ser Tyr Phe Ile Glu AlaThr Gly 1685 1690 1695 aat aag aac cac tta tgg gta cgt gct aaa tac caaaag gaa acg act 5136 Asn Lys Asn His Leu Trp Val Arg Ala Lys Tyr Gln LysGlu Thr Thr 1700 1705 1710 gat aag atc ttg ttc gac cgt act gat gag aaagat ccg cac ggt tgg 5184 Asp Lys Ile Leu Phe Asp Arg Thr Asp Glu Lys AspPro His Gly Trp 1715 1720 1725 ttt ctc agc gac gat cac aag acc ttt agtggt ctc tct tcc gca cag 5232 Phe Leu Ser Asp Asp His Lys Thr Phe Ser GlyLeu Ser Ser Ala Gln 1730 1735 1740 gca tta aag aac gac agt gaa ccg atggat ttc tct ggc gcc aat gct 5280 Ala Leu Lys Asn Asp Ser Glu Pro Met AspPhe Ser Gly Ala Asn Ala 1745 1750 1755 1760 ctc tat ttc tgg gaa ctg ttctat tac acg ccg atg atg atg gct cat 5328 Leu Tyr Phe Trp Glu Leu Phe TyrTyr Thr Pro Met Met Met Ala His 1765 1770 1775 cgt ttg ttg cag gaa cagaat ttt gat gcg gcg aac cat tgg ttc cgt 5376 Arg Leu Leu Gln Glu Gln AsnPhe Asp Ala Ala Asn His Trp Phe Arg 1780 1785 1790 tat gtc tgg agt ccatcc ggt tat atc gtt gat ggt aaa att gct atc 5424 Tyr Val Trp Ser Pro SerGly Tyr Ile Val Asp Gly Lys Ile Ala Ile 1795 1800 1805 tac cac tgg aacgtg cga ccg ctg gaa gaa gac acc agt tgg aat gca 5472 Tyr His Trp Asn ValArg Pro Leu Glu Glu Asp Thr Ser Trp Asn Ala 1810 1815 1820 caa caa ctggac tcc acc gat cca gat gct gta gcc caa gat gat ccg 5520 Gln Gln Leu AspSer Thr Asp Pro Asp Ala Val Ala Gln Asp Asp Pro 1825 1830 1835 1840 atgcac tac aag gtg gct acc ttt atg gcg acg ttg gat ctg cta atg 5568 Met HisTyr Lys Val Ala Thr Phe Met Ala Thr Leu Asp Leu Leu Met 1845 1850 1855gcc cgt ggt gat gct gct tac cgc cag tta gag cgt gat acg ttg gct 5616 AlaArg Gly Asp Ala Ala Tyr Arg Gln Leu Glu Arg Asp Thr Leu Ala 1860 18651870 gaa gct aaa atg tgg tat aca cag gcg ctt aat ctg ttg ggt gat gag5664 Glu Ala Lys Met Trp Tyr Thr Gln Ala Leu Asn Leu Leu Gly Asp Glu1875 1880 1885 cca caa gtg atg ctg agt acg act tgg gct aat cca aca ttgggt aat 5712 Pro Gln Val Met Leu Ser Thr Thr Trp Ala Asn Pro Thr Leu GlyAsn 1890 1895 1900 gct gct tca aaa acc aca cag cag gtt cgt cag caa gtgctt acc cag 5760 Ala Ala Ser Lys Thr Thr Gln Gln Val Arg Gln Gln Val LeuThr Gln 1905 1910 1915 1920 ttg cgt ctc aat agc agg gta aaa acc ccg ttgcta gga aca gcc aat 5808 Leu Arg Leu Asn Ser Arg Val Lys Thr Pro Leu LeuGly Thr Ala Asn 1925 1930 1935 tcc ctg acc gct tta ttc ctg ccg cag gaaaat agc aag ctc aaa ggc 5856 Ser Leu Thr Ala Leu Phe Leu Pro Gln Glu AsnSer Lys Leu Lys Gly 1940 1945 1950 tac tgg cgg aca ctg gcg cag cgt atgttt aat tta cgt cat aat ctg 5904 Tyr Trp Arg Thr Leu Ala Gln Arg Met PheAsn Leu Arg His Asn Leu 1955 1960 1965 tcg att gac ggc cag ccg ctc tccttg ccg ctg tat gct aaa ccg gct 5952 Ser Ile Asp Gly Gln Pro Leu Ser LeuPro Leu Tyr Ala Lys Pro Ala 1970 1975 1980 gat cca aaa gct tta ctg agtgcg gcg gtt tca gct tct caa ggg gga 6000 Asp Pro Lys Ala Leu Leu Ser AlaAla Val Ser Ala Ser Gln Gly Gly 1985 1990 1995 2000 gcc gac ttg ccg aaggcg ccg ctg act att cac cgc ttc cct caa atg 6048 Ala Asp Leu Pro Lys AlaPro Leu Thr Ile His Arg Phe Pro Gln Met 2005 2010 2015 cta gaa ggg gcacgg ggc ttg gtt aac cag ctt ata cag ttc ggt agt 6096 Leu Glu Gly Ala ArgGly Leu Val Asn Gln Leu Ile Gln Phe Gly Ser 2020 2025 2030 tca cta ttgggg tac agt gag cgt cag gat gcg gaa gct atg agt caa 6144 Ser Leu Leu GlyTyr Ser Glu Arg Gln Asp Ala Glu Ala Met Ser Gln 2035 2040 2045 cta ctgcaa acc caa gcc agc gag tta ata ctg acc agt att cgt atg 6192 Leu Leu GlnThr Gln Ala Ser Glu Leu Ile Leu Thr Ser Ile Arg Met 2050 2055 2060 caggat aac caa ttg gca gag ctg gat tcg gaa aaa acc gcc ttg caa 6240 Gln AspAsn Gln Leu Ala Glu Leu Asp Ser Glu Lys Thr Ala Leu Gln 2065 2070 20752080 gtc tct tta gct gga gtg caa caa cgg ttt gac agc tat agc caa ctg6288 Val Ser Leu Ala Gly Val Gln Gln Arg Phe Asp Ser Tyr Ser Gln Leu2085 2090 2095 tat gag gag aac atc aac gca ggt gag cag cga gcg ctg gcgtta cgc 6336 Tyr Glu Glu Asn Ile Asn Ala Gly Glu Gln Arg Ala Leu Ala LeuArg 2100 2105 2110 tca gaa tct gct att gag tct cag gga gcg cag att tcccgt atg gca 6384 Ser Glu Ser Ala Ile Glu Ser Gln Gly Ala Gln Ile Ser ArgMet Ala 2115 2120 2125 ggc gcg ggt gtt gat atg gca cca aat atc ttc ggcctg gct gat ggc 6432 Gly Ala Gly Val Asp Met Ala Pro Asn Ile Phe Gly LeuAla Asp Gly 2130 2135 2140 ggc atg cat tat ggt gct att gcc tat gcc atcgct gac ggt att gag 6480 Gly Met His Tyr Gly Ala Ile Ala Tyr Ala Ile AlaAsp Gly Ile Glu 2145 2150 2155 2160 ttg agt gct tct gcc aag atg gtt gatgcg gag aaa gtt gct cag tcg 6528 Leu Ser Ala Ser Ala Lys Met Val Asp AlaGlu Lys Val Ala Gln Ser 2165 2170 2175 gaa ata tat cgc cgt cgc cgt caagaa tgg aaa att cag cgt gac aac 6576 Glu Ile Tyr Arg Arg Arg Arg Gln GluTrp Lys Ile Gln Arg Asp Asn 2180 2185 2190 gca caa gcg gag att aac cagtta aac gcg caa ctg gaa tca ctg tct 6624 Ala Gln Ala Glu Ile Asn Gln LeuAsn Ala Gln Leu Glu Ser Leu Ser 2195 2200 2205 att cgc cgt gaa gcc gctgaa atg caa aaa gag tac ctg aaa acc cag 6672 Ile Arg Arg Glu Ala Ala GluMet Gln Lys Glu Tyr Leu Lys Thr Gln 2210 2215 2220 caa gct cag gcg caggca caa ctt act ttc tta aga agc aaa ttc agt 6720 Gln Ala Gln Ala Gln AlaGln Leu Thr Phe Leu Arg Ser Lys Phe Ser 2225 2230 2235 2240 aat caa gcgtta tat agt tgg tta cga ggg cgt ttg tca ggt att tat 6768 Asn Gln Ala LeuTyr Ser Trp Leu Arg Gly Arg Leu Ser Gly Ile Tyr 2245 2250 2255 ttc cagttc tat gac ttg gcc gta tca cgt tgc ctg atg gca gag caa 6816 Phe Gln PheTyr Asp Leu Ala Val Ser Arg Cys Leu Met Ala Glu Gln 2260 2265 2270 tcctat caa tgg gaa gct aat gat aat tcc att agc ttt gtc aaa ccg 6864 Ser TyrGln Trp Glu Ala Asn Asp Asn Ser Ile Ser Phe Val Lys Pro 2275 2280 2285ggt gca tgg caa gga act tac gcc ggc tta ttg tgt gga gaa gct ttg 6912 GlyAla Trp Gln Gly Thr Tyr Ala Gly Leu Leu Cys Gly Glu Ala Leu 2290 22952300 ata caa aat ctg gca caa atg gaa gag gca tat ctg aaa tgg gaa tct6960 Ile Gln Asn Leu Ala Gln Met Glu Glu Ala Tyr Leu Lys Trp Glu Ser2305 2310 2315 2320 cgc gct ttg gaa gta gaa cgc acg gtt tca ttg gca gtggtt tat gat 7008 Arg Ala Leu Glu Val Glu Arg Thr Val Ser Leu Ala Val ValTyr Asp 2325 2330 2335 tca ctg gaa ggt aat gat cgt ttt aat tta gcg gaacaa ata cct gca 7056 Ser Leu Glu Gly Asn Asp Arg Phe Asn Leu Ala Glu GlnIle Pro Ala 2340 2345 2350 tta ttg gat aag ggg gag gga aca gca gga actaaa gaa aat ggg tta 7104 Leu Leu Asp Lys Gly Glu Gly Thr Ala Gly Thr LysGlu Asn Gly Leu 2355 2360 2365 tca ttg gct aat gct atc ctg tca gct tcggtc aaa ttg tcc gac ttg 7152 Ser Leu Ala Asn Ala Ile Leu Ser Ala Ser ValLys Leu Ser Asp Leu 2370 2375 2380 aaa ctg gga acg gat tat cca gac agtatc gtt ggt agc aac aag gtt 7200 Lys Leu Gly Thr Asp Tyr Pro Asp Ser IleVal Gly Ser Asn Lys Val 2385 2390 2395 2400 cgt cgt att aag caa atc agtgtt tcg cta cct gca ttg gtt ggg cct 7248 Arg Arg Ile Lys Gln Ile Ser ValSer Leu Pro Ala Leu Val Gly Pro 2405 2410 2415 tat cag gat gtt cag gctatg ctc agc tat ggt ggc agt act caa ttg 7296 Tyr Gln Asp Val Gln Ala MetLeu Ser Tyr Gly Gly Ser Thr Gln Leu 2420 2425 2430 ccg aaa ggt tgt tcagcg ttg gct gtg tct cat ggt acc aat gat agt 7344 Pro Lys Gly Cys Ser AlaLeu Ala Val Ser His Gly Thr Asn Asp Ser 2435 2440 2445 ggt cag ttc cagttg gat ttc aat gac ggc aaa tac ctg cca ttt gaa 7392 Gly Gln Phe Gln LeuAsp Phe Asn Asp Gly Lys Tyr Leu Pro Phe Glu 2450 2455 2460 ggt att gctctt gat gat cag ggt aca ctg aat ctt caa ttt ccg aat 7440 Gly Ile Ala LeuAsp Asp Gln Gly Thr Leu Asn Leu Gln Phe Pro Asn 2465 2470 2475 2480 gctacc gac aag cag aaa gca ata ttg caa act atg agc gat att att 7488 Ala ThrAsp Lys Gln Lys Ala Ile Leu Gln Thr Met Ser Asp Ile Ile 2485 2490 2495ttg cat att cgt tat acc atc cgt taa 7515 Leu His Ile Arg Tyr Thr Ile Arg2500 3 7577 DNA Artificial Sequence CDS (3)..(7553) Description ofArtificial Sequencehemicot tcdA 3 cc atg gct aac gag tcc gtc aag gag atccca gac gtc ctc aag tcc 47 Met Ala Asn Glu Ser Val Lys Glu Ile Pro AspVal Leu Lys Ser 1 5 10 15 caa tgc ggt ttc aac tgc ctc act gac atc tcccac agc tcc ttc aac 95 Gln Cys Gly Phe Asn Cys Leu Thr Asp Ile Ser HisSer Ser Phe Asn 20 25 30 gag ttc aga caa caa gtc tct gag cac ctc tcc tggtcc gag acc cat 143 Glu Phe Arg Gln Gln Val Ser Glu His Leu Ser Trp SerGlu Thr His 35 40 45 gac ctc tac cat gac gct cag caa gct cag aag gac aacagg ctc tac 191 Asp Leu Tyr His Asp Ala Gln Gln Ala Gln Lys Asp Asn ArgLeu Tyr 50 55 60 gag gct agg atc ctc aag agg gct aac cca caa ctc cag aacgct gtc 239 Glu Ala Arg Ile Leu Lys Arg Ala Asn Pro Gln Leu Gln Asn AlaVal 65 70 75 cac ctc gcc atc ttg gct cca aac gct gag ttg att ggt tac aacaac 287 His Leu Ala Ile Leu Ala Pro Asn Ala Glu Leu Ile Gly Tyr Asn Asn80 85 90 95 cag ttc tct ggc aga gct agc cag tac gtg gct cct ggt aca gtctcc 335 Gln Phe Ser Gly Arg Ala Ser Gln Tyr Val Ala Pro Gly Thr Val Ser100 105 110 tcc atg ttc agc cca gcc gct tac ctc act gag ttg tac cgc gaggct 383 Ser Met Phe Ser Pro Ala Ala Tyr Leu Thr Glu Leu Tyr Arg Glu Ala115 120 125 agg aac ctt cat gct tct gac tcc gtc tac tac ttg gac aca cgcaga 431 Arg Asn Leu His Ala Ser Asp Ser Val Tyr Tyr Leu Asp Thr Arg Arg130 135 140 cca gac ctc aag agc atg gcc ctc agc caa cag aac atg gac attgag 479 Pro Asp Leu Lys Ser Met Ala Leu Ser Gln Gln Asn Met Asp Ile Glu145 150 155 ttg tcc acc ctc tcc ttg agc aac gag ctt ctc ttg gag tcc atcaag 527 Leu Ser Thr Leu Ser Leu Ser Asn Glu Leu Leu Leu Glu Ser Ile Lys160 165 170 175 act gag agc aag ttg gag aac tac acc aag gtc atg gag atgctc tcc 575 Thr Glu Ser Lys Leu Glu Asn Tyr Thr Lys Val Met Glu Met LeuSer 180 185 190 acc ttc aga cca agc ggt gca act cca tac cat gat gcc tacgag aac 623 Thr Phe Arg Pro Ser Gly Ala Thr Pro Tyr His Asp Ala Tyr GluAsn 195 200 205 gtc agg gag gtc atc caa ctt caa gac cct ggt ctt gag caactc aac 671 Val Arg Glu Val Ile Gln Leu Gln Asp Pro Gly Leu Glu Gln LeuAsn 210 215 220 gct tct cca gcc att gct ggt ttg atg cac cag gca tcc ttgctc ggt 719 Ala Ser Pro Ala Ile Ala Gly Leu Met His Gln Ala Ser Leu LeuGly 225 230 235 atc aac gcc tcc atc tct cct gag ttg ttc aac atc ttg actgag gag 767 Ile Asn Ala Ser Ile Ser Pro Glu Leu Phe Asn Ile Leu Thr GluGlu 240 245 250 255 atc act gag ggc aac gct gag gag ttg tac aag aag aacttc ggc aac 815 Ile Thr Glu Gly Asn Ala Glu Glu Leu Tyr Lys Lys Asn PheGly Asn 260 265 270 att gag cca gcc tct ctt gca atg cct gag tac ctc aagagg tac tac 863 Ile Glu Pro Ala Ser Leu Ala Met Pro Glu Tyr Leu Lys ArgTyr Tyr 275 280 285 aac ttg tct gat gag gag ctt tct caa ttc att ggc aaggct tcc aac 911 Asn Leu Ser Asp Glu Glu Leu Ser Gln Phe Ile Gly Lys AlaSer Asn 290 295 300 ttc ggt caa cag gag tac agc aac aac cag ctc atc actcca gtt gtg 959 Phe Gly Gln Gln Glu Tyr Ser Asn Asn Gln Leu Ile Thr ProVal Val 305 310 315 aac tcc tct gat ggc act gtg aag gtc tac cgc atc acacgt gag tac 1007 Asn Ser Ser Asp Gly Thr Val Lys Val Tyr Arg Ile Thr ArgGlu Tyr 320 325 330 335 acc aca aac gcc tac caa atg gat gtt gag ttg ttccca ttc ggt ggt 1055 Thr Thr Asn Ala Tyr Gln Met Asp Val Glu Leu Phe ProPhe Gly Gly 340 345 350 gag aac tac aga ctt gac tac aag ttc aag aac ttctac aac gcc tcc 1103 Glu Asn Tyr Arg Leu Asp Tyr Lys Phe Lys Asn Phe TyrAsn Ala Ser 355 360 365 tac ctc tcc atc aag ttg aac gac aag agg gag cttgtc agg act gag 1151 Tyr Leu Ser Ile Lys Leu Asn Asp Lys Arg Glu Leu ValArg Thr Glu 370 375 380 ggt gct cct caa gtg aac att gag tac tct gcc aacatc acc ctc aac 1199 Gly Ala Pro Gln Val Asn Ile Glu Tyr Ser Ala Asn IleThr Leu Asn 385 390 395 aca gct gac atc tct caa cca ttc gag att ggt ttgacc aga gtc ctt 1247 Thr Ala Asp Ile Ser Gln Pro Phe Glu Ile Gly Leu ThrArg Val Leu 400 405 410 415 ccc tct ggc tcc tgg gcc tac gct gca gcc aagttc act gtt gag gag 1295 Pro Ser Gly Ser Trp Ala Tyr Ala Ala Ala Lys PheThr Val Glu Glu 420 425 430 tac aac cag tac tct ttc ctc ttg aag ctc aacaag gca att cgt ctc 1343 Tyr Asn Gln Tyr Ser Phe Leu Leu Lys Leu Asn LysAla Ile Arg Leu 435 440 445 agc aga gcc act gag ttg tct ccc acc atc ttggag ggc att gtg agg 1391 Ser Arg Ala Thr Glu Leu Ser Pro Thr Ile Leu GluGly Ile Val Arg 450 455 460 tct gtc aac ctt caa ctt gac atc aac act gatgtg ctt ggc aag gtc 1439 Ser Val Asn Leu Gln Leu Asp Ile Asn Thr Asp ValLeu Gly Lys Val 465 470 475 ttc ctc acc aag tac tac atg caa cgc tac gccatc cat gct gag act 1487 Phe Leu Thr Lys Tyr Tyr Met Gln Arg Tyr Ala IleHis Ala Glu Thr 480 485 490 495 gca ctc atc ctc tgc aac gca ccc atc tctcaa cgc tcc tac gac aac 1535 Ala Leu Ile Leu Cys Asn Ala Pro Ile Ser GlnArg Ser Tyr Asp Asn 500 505 510 cag cct tcc cag ttc gac agg ctc ttc aacact cct ctc ttg aac ggc 1583 Gln Pro Ser Gln Phe Asp Arg Leu Phe Asn ThrPro Leu Leu Asn Gly 515 520 525 cag tac ttc tcc act ggt gat gag gag attgac ctc aac tct ggc tcc 1631 Gln Tyr Phe Ser Thr Gly Asp Glu Glu Ile AspLeu Asn Ser Gly Ser 530 535 540 aca ggt gac tgg aga aag acc atc ttg aagagg gcc ttc aac att gat 1679 Thr Gly Asp Trp Arg Lys Thr Ile Leu Lys ArgAla Phe Asn Ile Asp 545 550 555 gat gtc tct ctc ttc cgt ctc ttg aag atcaca gat cac gac aac aag 1727 Asp Val Ser Leu Phe Arg Leu Leu Lys Ile ThrAsp His Asp Asn Lys 560 565 570 575 gat ggc aag atc aag aac aac ttg aagaac ctt tcc aac ctc tac att 1775 Asp Gly Lys Ile Lys Asn Asn Leu Lys AsnLeu Ser Asn Leu Tyr Ile 580 585 590 ggc aag ttg ctt gca gac atc cac caactc acc att gat gag ttg gac 1823 Gly Lys Leu Leu Ala Asp Ile His Gln LeuThr Ile Asp Glu Leu Asp 595 600 605 ctc ttg ctc att gca gtc ggt gag ggcaag acc aac ctc tct gca atc 1871 Leu Leu Leu Ile Ala Val Gly Glu Gly LysThr Asn Leu Ser Ala Ile 610 615 620 tct gac aag cag ttg gca acc ctc atcagg aag ttg aac acc atc acc 1919 Ser Asp Lys Gln Leu Ala Thr Leu Ile ArgLys Leu Asn Thr Ile Thr 625 630 635 tcc tgg ctt cac acc cag aag tgg tctgtc ttc caa ctc ttc atc atg 1967 Ser Trp Leu His Thr Gln Lys Trp Ser ValPhe Gln Leu Phe Ile Met 640 645 650 655 acc agc acc tcc tac aac aag accctc act cct gag atc aag aac ctc 2015 Thr Ser Thr Ser Tyr Asn Lys Thr LeuThr Pro Glu Ile Lys Asn Leu 660 665 670 ttg gac aca gtc tac cac ggt ctccaa ggc ttc gac aag gac aag gct 2063 Leu Asp Thr Val Tyr His Gly Leu GlnGly Phe Asp Lys Asp Lys Ala 675 680 685 gac ttg ctt cat gtc atg gct ccctac att gca gcc acc ctc caa ctc 2111 Asp Leu Leu His Val Met Ala Pro TyrIle Ala Ala Thr Leu Gln Leu 690 695 700 tcc tct gag aac gtg gct cac tctgtc ttg ctc tgg gct gac aag ctc 2159 Ser Ser Glu Asn Val Ala His Ser ValLeu Leu Trp Ala Asp Lys Leu 705 710 715 caa cct ggt gat ggt gcc atg actgct gag aag ttc tgg gac tgg ctc 2207 Gln Pro Gly Asp Gly Ala Met Thr AlaGlu Lys Phe Trp Asp Trp Leu 720 725 730 735 aac acc aag tac aca cca ggctcc tct gag gct gtt gag act caa gag 2255 Asn Thr Lys Tyr Thr Pro Gly SerSer Glu Ala Val Glu Thr Gln Glu 740 745 750 cac att gtg caa tac tgc caggct ctt gca cag ttg gag atg gtc tac 2303 His Ile Val Gln Tyr Cys Gln AlaLeu Ala Gln Leu Glu Met Val Tyr 755 760 765 cac tcc act ggc atc aac gagaac gct ttc aga ctc ttc gtc acc aag 2351 His Ser Thr Gly Ile Asn Glu AsnAla Phe Arg Leu Phe Val Thr Lys 770 775 780 cct gag atg ttc ggt gct gccaca ggt gct gca cct gct cat gat gct 2399 Pro Glu Met Phe Gly Ala Ala ThrGly Ala Ala Pro Ala His Asp Ala 785 790 795 ctc tcc ctc atc atg ttg accagg ttc gct gac tgg gtc aac gct ctt 2447 Leu Ser Leu Ile Met Leu Thr ArgPhe Ala Asp Trp Val Asn Ala Leu 800 805 810 815 ggt gag aag gct tcc tctgtc ttg gct gcc ttc gag gcc aac tcc ctc 2495 Gly Glu Lys Ala Ser Ser ValLeu Ala Ala Phe Glu Ala Asn Ser Leu 820 825 830 act gct gag caa ctt gctgat gcc atg aac ctt gat gcc aac ctc ttg 2543 Thr Ala Glu Gln Leu Ala AspAla Met Asn Leu Asp Ala Asn Leu Leu 835 840 845 ctc caa gct tcc att caagct cag aac cac caa cac ctc cca cct gtc 2591 Leu Gln Ala Ser Ile Gln AlaGln Asn His Gln His Leu Pro Pro Val 850 855 860 act cca gag aac gct ttctcc tgc tgg acc tcc atc aac acc atc ctc 2639 Thr Pro Glu Asn Ala Phe SerCys Trp Thr Ser Ile Asn Thr Ile Leu 865 870 875 caa tgg gtc aac gtg gctcag caa ctc aac gtg gct cca caa ggt gtc 2687 Gln Trp Val Asn Val Ala GlnGln Leu Asn Val Ala Pro Gln Gly Val 880 885 890 895 tct gct ttg gtc ggtctt gac tac atc cag tcc atg aag gag aca cca 2735 Ser Ala Leu Val Gly LeuAsp Tyr Ile Gln Ser Met Lys Glu Thr Pro 900 905 910 acc tac gct caa tgggag aac gca gct ggt gtc ttg act gct ggt ctc 2783 Thr Tyr Ala Gln Trp GluAsn Ala Ala Gly Val Leu Thr Ala Gly Leu 915 920 925 aac tcc caa cag gccaac acc ctc cat gct ttc ttg gat gag tct cgc 2831 Asn Ser Gln Gln Ala AsnThr Leu His Ala Phe Leu Asp Glu Ser Arg 930 935 940 tct gct gcc ctc tccacc tac tac atc agg caa gtc gcc aag gca gct 2879 Ser Ala Ala Leu Ser ThrTyr Tyr Ile Arg Gln Val Ala Lys Ala Ala 945 950 955 gct gcc atc aag tctcgc gat gac ctc tac caa tac ctc ctc att gac 2927 Ala Ala Ile Lys Ser ArgAsp Asp Leu Tyr Gln Tyr Leu Leu Ile Asp 960 965 970 975 aac cag gtc tctgct gcc atc aag acc acc agg atc gct gag gcc atc 2975 Asn Gln Val Ser AlaAla Ile Lys Thr Thr Arg Ile Ala Glu Ala Ile 980 985 990 gct tcc atc caactc tac gtc aac cgc gct ctt gag aac gtt gag gag 3023 Ala Ser Ile Gln LeuTyr Val Asn Arg Ala Leu Glu Asn Val Glu Glu 995 1000 1005 aac gcc aactct ggt gtc atc tct cgc caa ttc ttc atc gac tgg gac 3071 Asn Ala Asn SerGly Val Ile Ser Arg Gln Phe Phe Ile Asp Trp Asp 1010 1015 1020 aag tacaac aag agg tac tcc acc tgg gct ggt gtc tct caa ctt gtc 3119 Lys Tyr AsnLys Arg Tyr Ser Thr Trp Ala Gly Val Ser Gln Leu Val 1025 1030 1035 tactac cca gag aac tac att gac cca acc atg agg att ggt cag acc 3167 Tyr TyrPro Glu Asn Tyr Ile Asp Pro Thr Met Arg Ile Gly Gln Thr 1040 1045 10501055 aag atg atg gat gct ctc ttg caa tct gtc tcc caa agc caa ctc aac3215 Lys Met Met Asp Ala Leu Leu Gln Ser Val Ser Gln Ser Gln Leu Asn1060 1065 1070 gct gac act gtg gag gat gcc ttc atg agc tac ctc acc tccttc gag 3263 Ala Asp Thr Val Glu Asp Ala Phe Met Ser Tyr Leu Thr Ser PheGlu 1075 1080 1085 caa gtt gcc aac ctc aag gtc atc tct gct tac cat gacaac atc aac 3311 Gln Val Ala Asn Leu Lys Val Ile Ser Ala Tyr His Asp AsnIle Asn 1090 1095 1100 aac gac caa ggt ctc acc tac ttc att ggt ctc tctgag act gat gct 3359 Asn Asp Gln Gly Leu Thr Tyr Phe Ile Gly Leu Ser GluThr Asp Ala 1105 1110 1115 ggt gag tac tac tgg aga tcc gtg gac cac agcaag ttc aac gat ggc 3407 Gly Glu Tyr Tyr Trp Arg Ser Val Asp His Ser LysPhe Asn Asp Gly 1120 1125 1130 1135 aag ttc gct gca aac gct tgg tct gagtgg cac aag att gac tgc cct 3455 Lys Phe Ala Ala Asn Ala Trp Ser Glu TrpHis Lys Ile Asp Cys Pro 1140 1145 1150 atc aac cca tac aag tcc acc atcaga cct gtc atc tac aag agc cgc 3503 Ile Asn Pro Tyr Lys Ser Thr Ile ArgPro Val Ile Tyr Lys Ser Arg 1155 1160 1165 ctc tac ttg ctc tgg ctt gagcag aag gag atc acc aag caa act ggc 3551 Leu Tyr Leu Leu Trp Leu Glu GlnLys Glu Ile Thr Lys Gln Thr Gly 1170 1175 1180 aac tcc aag gat ggt taccaa act gag act gac tac cgc tac gag ttg 3599 Asn Ser Lys Asp Gly Tyr GlnThr Glu Thr Asp Tyr Arg Tyr Glu Leu 1185 1190 1195 aag ttg gct cac atccgc tac gat ggt acc tgg aac act cca atc acc 3647 Lys Leu Ala His Ile ArgTyr Asp Gly Thr Trp Asn Thr Pro Ile Thr 1200 1205 1210 1215 ttc gat gtcaac aag aag atc agc gag ttg aag ttg gag aag aac cgt 3695 Phe Asp Val AsnLys Lys Ile Ser Glu Leu Lys Leu Glu Lys Asn Arg 1220 1225 1230 gct cctggt ctc tac tgc gct ggt tac caa ggt gag gac acc ctc ttg 3743 Ala Pro GlyLeu Tyr Cys Ala Gly Tyr Gln Gly Glu Asp Thr Leu Leu 1235 1240 1245 gtcatg ttc tac aac cag caa gac acc ctt gac tcc tac aag aac gct 3791 Val MetPhe Tyr Asn Gln Gln Asp Thr Leu Asp Ser Tyr Lys Asn Ala 1250 1255 1260tcc atg caa ggt ctc tac atc ttc gct gac atg gct tcc aag gac atg 3839 SerMet Gln Gly Leu Tyr Ile Phe Ala Asp Met Ala Ser Lys Asp Met 1265 12701275 act cca gag caa agc aac gtc tac cgt gac aac tcc tac caa cag ttc3887 Thr Pro Glu Gln Ser Asn Val Tyr Arg Asp Asn Ser Tyr Gln Gln Phe1280 1285 1290 1295 gac acc aac aac gtc agg cgt gtc aac aac aga tac gctgag gac tac 3935 Asp Thr Asn Asn Val Arg Arg Val Asn Asn Arg Tyr Ala GluAsp Tyr 1300 1305 1310 gag atc cca agc tct gtc agc tct cgc aag gac tacggc tgg ggt gac 3983 Glu Ile Pro Ser Ser Val Ser Ser Arg Lys Asp Tyr GlyTrp Gly Asp 1315 1320 1325 tac tac ctc agc atg gtg tac aac ggt gac atccca acc atc aac tac 4031 Tyr Tyr Leu Ser Met Val Tyr Asn Gly Asp Ile ProThr Ile Asn Tyr 1330 1335 1340 aag gct gcc tct tcc gac ctc aaa atc tacatc agc cca aag ctc agg 4079 Lys Ala Ala Ser Ser Asp Leu Lys Ile Tyr IleSer Pro Lys Leu Arg 1345 1350 1355 atc atc cac aac ggc tac gag ggt cagaag agg aac cag tgc aac ttg 4127 Ile Ile His Asn Gly Tyr Glu Gly Gln LysArg Asn Gln Cys Asn Leu 1360 1365 1370 1375 atg aac aag tac ggc aag ttgggt gac aag ttc att gtc tac acc tct 4175 Met Asn Lys Tyr Gly Lys Leu GlyAsp Lys Phe Ile Val Tyr Thr Ser 1380 1385 1390 ctt ggt gtc aac cca aacaac agc tcc aac aag ctc atg ttc tac cca 4223 Leu Gly Val Asn Pro Asn AsnSer Ser Asn Lys Leu Met Phe Tyr Pro 1395 1400 1405 gtc tac caa tac tctggc aac acc tct ggt ctc aac cag ggt aga ctc 4271 Val Tyr Gln Tyr Ser GlyAsn Thr Ser Gly Leu Asn Gln Gly Arg Leu 1410 1415 1420 ttg ttc cac agggac acc acc tac cca agc aag gtg gag gct tgg att 4319 Leu Phe His Arg AspThr Thr Tyr Pro Ser Lys Val Glu Ala Trp Ile 1425 1430 1435 cct ggt gccaag agg tcc ctc acc aac cag aac gct gcc att ggt gat 4367 Pro Gly Ala LysArg Ser Leu Thr Asn Gln Asn Ala Ala Ile Gly Asp 1440 1445 1450 1455 gactac gcc aca gac tcc ctc aac aag cct gat gac ctc aag cag tac 4415 Asp TyrAla Thr Asp Ser Leu Asn Lys Pro Asp Asp Leu Lys Gln Tyr 1460 1465 1470atc ttc atg act gac tcc aag ggc aca gcc act gat gtc tct ggt cca 4463 IlePhe Met Thr Asp Ser Lys Gly Thr Ala Thr Asp Val Ser Gly Pro 1475 14801485 gtg gag atc aac act gca atc agc cca gcc aag gtc caa atc att gtc4511 Val Glu Ile Asn Thr Ala Ile Ser Pro Ala Lys Val Gln Ile Ile Val1490 1495 1500 aag gct ggt ggc aag gag caa acc ttc aca gct gac aag gatgtc tcc 4559 Lys Ala Gly Gly Lys Glu Gln Thr Phe Thr Ala Asp Lys Asp ValSer 1505 1510 1515 atc cag cca agc cca tcc ttc gat gag atg aac tac caattc aac gct 4607 Ile Gln Pro Ser Pro Ser Phe Asp Glu Met Asn Tyr Gln PheAsn Ala 1520 1525 1530 1535 ctt gag att gat ggt tct ggc ctc aac ttc atcaac aac tct gct tcc 4655 Leu Glu Ile Asp Gly Ser Gly Leu Asn Phe Ile AsnAsn Ser Ala Ser 1540 1545 1550 att gat gtc acc ttc act gcc ttc gct gaggat ggc cgc aag ttg ggt 4703 Ile Asp Val Thr Phe Thr Ala Phe Ala Glu AspGly Arg Lys Leu Gly 1555 1560 1565 tac gag agc ttc tcc atc cca gtc accctt aag gtt tcc act gac aac 4751 Tyr Glu Ser Phe Ser Ile Pro Val Thr LeuLys Val Ser Thr Asp Asn 1570 1575 1580 gca ctc acc ctt cat cac aac gagaac ggt gct cag tac atg caa tgg 4799 Ala Leu Thr Leu His His Asn Glu AsnGly Ala Gln Tyr Met Gln Trp 1585 1590 1595 caa agc tac cgc acc agg ttgaac acc ctc ttc gca agg caa ctt gtg 4847 Gln Ser Tyr Arg Thr Arg Leu AsnThr Leu Phe Ala Arg Gln Leu Val 1600 1605 1610 1615 gcc cgt gcc acc acaggc att gac acc atc ctc agc atg gag acc cag 4895 Ala Arg Ala Thr Thr GlyIle Asp Thr Ile Leu Ser Met Glu Thr Gln 1620 1625 1630 aac atc caa gagcca cag ttg ggc aag ggt ttc tac gcc acc ttc gtc 4943 Asn Ile Gln Glu ProGln Leu Gly Lys Gly Phe Tyr Ala Thr Phe Val 1635 1640 1645 atc cca ccttac aac ctc agc act cat ggt gat gag agg tgg ttc aag 4991 Ile Pro Pro TyrAsn Leu Ser Thr His Gly Asp Glu Arg Trp Phe Lys 1650 1655 1660 ctc tacatc aag cac gtg gtt gac aac aac tcc cac atc atc tac tct 5039 Leu Tyr IleLys His Val Val Asp Asn Asn Ser His Ile Ile Tyr Ser 1665 1670 1675 ggtcaa ctc act gac acc aac atc aac atc acc ctc ttc atc cca ctt 5087 Gly GlnLeu Thr Asp Thr Asn Ile Asn Ile Thr Leu Phe Ile Pro Leu 1680 1685 16901695 gac gat gtc cca ctc aac cag gac tac cat gcc aag gtc tac atg acc5135 Asp Asp Val Pro Leu Asn Gln Asp Tyr His Ala Lys Val Tyr Met Thr1700 1705 1710 ttc aag aag tct cca tct gat ggc acc tgg tgg ggt cca cacttc gtc 5183 Phe Lys Lys Ser Pro Ser Asp Gly Thr Trp Trp Gly Pro His PheVal 1715 1720 1725 cgt gat gac aag ggc atc gtc acc atc aac cca aag tccatc ctc acc 5231 Arg Asp Asp Lys Gly Ile Val Thr Ile Asn Pro Lys Ser IleLeu Thr 1730 1735 1740 cac ttc gag tct gtc aac gtt ctc aac aac atc tcctct gag cca atg 5279 His Phe Glu Ser Val Asn Val Leu Asn Asn Ile Ser SerGlu Pro Met 1745 1750 1755 gac ttc tct ggt gcc aac tcc ctc tac ttc tgggag ttg ttc tac tac 5327 Asp Phe Ser Gly Ala Asn Ser Leu Tyr Phe Trp GluLeu Phe Tyr Tyr 1760 1765 1770 1775 aca cca atg ctt gtg gct caa agg ttgctc cat gag cag aac ttc gat 5375 Thr Pro Met Leu Val Ala Gln Arg Leu LeuHis Glu Gln Asn Phe Asp 1780 1785 1790 gag gcc aac agg tgg ctc aag tacgtc tgg agc cca tct ggt tac att 5423 Glu Ala Asn Arg Trp Leu Lys Tyr ValTrp Ser Pro Ser Gly Tyr Ile 1795 1800 1805 gtg cat ggt caa atc cag aactac caa tgg aac gtc agg cca ttg ctt 5471 Val His Gly Gln Ile Gln Asn TyrGln Trp Asn Val Arg Pro Leu Leu 1810 1815 1820 gag gac acc tcc tgg aactct gac cca ctt gac tct gtg gac cct gat 5519 Glu Asp Thr Ser Trp Asn SerAsp Pro Leu Asp Ser Val Asp Pro Asp 1825 1830 1835 gct gtg gct caa catgac cca atg cac tac aag gtc tcc acc ttc atg 5567 Ala Val Ala Gln His AspPro Met His Tyr Lys Val Ser Thr Phe Met 1840 1845 1850 1855 agg acc ttggac ctc ttg att gcc aga ggt gac cat gct tac cgc caa 5615 Arg Thr Leu AspLeu Leu Ile Ala Arg Gly Asp His Ala Tyr Arg Gln 1860 1865 1870 ttg gagagg gac acc ctc aac gag gca aag atg tgg tac atg caa gct 5663 Leu Glu ArgAsp Thr Leu Asn Glu Ala Lys Met Trp Tyr Met Gln Ala 1875 1880 1885 ctccac ctc ttg ggt gac aag cca tac ctc cca ctc agc acc act tgg 5711 Leu HisLeu Leu Gly Asp Lys Pro Tyr Leu Pro Leu Ser Thr Thr Trp 1890 1895 1900tcc gac cca agg ttg gac cgt gct gct gac atc acc act cag aac gct 5759 SerAsp Pro Arg Leu Asp Arg Ala Ala Asp Ile Thr Thr Gln Asn Ala 1905 19101915 cat gac tct gcc att gtt gct ctc agg cag aac atc cca act cct gct5807 His Asp Ser Ala Ile Val Ala Leu Arg Gln Asn Ile Pro Thr Pro Ala1920 1925 1930 1935 cca ctc tcc ctc aga tct gct aac acc ctc act gac ttgttc ctc cca 5855 Pro Leu Ser Leu Arg Ser Ala Asn Thr Leu Thr Asp Leu PheLeu Pro 1940 1945 1950 cag atc aac gag gtc atg atg aac tac tgg caa accttg gct caa agg 5903 Gln Ile Asn Glu Val Met Met Asn Tyr Trp Gln Thr LeuAla Gln Arg 1955 1960 1965 gtc tac aac ctc aga cac aac ctc tcc att gatggt caa cca ctc tac 5951 Val Tyr Asn Leu Arg His Asn Leu Ser Ile Asp GlyGln Pro Leu Tyr 1970 1975 1980 ctc cca atc tac gcc aca cca gct gac ccaaag gct ctt ctc tct gct 5999 Leu Pro Ile Tyr Ala Thr Pro Ala Asp Pro LysAla Leu Leu Ser Ala 1985 1990 1995 gct gtg gct acc agc caa ggt ggt ggcaag ctc cca gag tcc ttc atg 6047 Ala Val Ala Thr Ser Gln Gly Gly Gly LysLeu Pro Glu Ser Phe Met 2000 2005 2010 2015 tcc ctc tgg agg ttc cca cacatg ttg gag aac gcc cgt ggc atg gtc 6095 Ser Leu Trp Arg Phe Pro His MetLeu Glu Asn Ala Arg Gly Met Val 2020 2025 2030 tcc caa ctc acc cag ttcggt tcc acc ctc cag aac atc att gag agg 6143 Ser Gln Leu Thr Gln Phe GlySer Thr Leu Gln Asn Ile Ile Glu Arg 2035 2040 2045 caa gat gct gag gctctc aac gct ttg ctc cag aac cag gca gct gag 6191 Gln Asp Ala Glu Ala LeuAsn Ala Leu Leu Gln Asn Gln Ala Ala Glu 2050 2055 2060 ttg atc ctc accaac ttg tcc atc caa gac aag acc att gag gag ctt 6239 Leu Ile Leu Thr AsnLeu Ser Ile Gln Asp Lys Thr Ile Glu Glu Leu 2065 2070 2075 gat gct gagaag aca gtc ctt gag aag agc aag gct ggt gcc caa tct 6287 Asp Ala Glu LysThr Val Leu Glu Lys Ser Lys Ala Gly Ala Gln Ser 2080 2085 2090 2095 cgcttc gac tcc tac ggc aag ctc tac gat gag aac atc aac gct ggt 6335 Arg PheAsp Ser Tyr Gly Lys Leu Tyr Asp Glu Asn Ile Asn Ala Gly 2100 2105 2110gag aac cag gcc atg acc ctc agg gct tcc gca gct ggt ctc acc act 6383 GluAsn Gln Ala Met Thr Leu Arg Ala Ser Ala Ala Gly Leu Thr Thr 2115 21202125 gct gtc caa gcc tct cgc ttg gct ggt gca gct gct gac ctc gtt cca6431 Ala Val Gln Ala Ser Arg Leu Ala Gly Ala Ala Ala Asp Leu Val Pro2130 2135 2140 aac atc ttc ggt ttc gct ggt ggt ggc tcc aga tgg ggt gccatt gct 6479 Asn Ile Phe Gly Phe Ala Gly Gly Gly Ser Arg Trp Gly Ala IleAla 2145 2150 2155 gag gct acc ggt tac gtc atg gag ttc tct gcc aac gtcatg aac act 6527 Glu Ala Thr Gly Tyr Val Met Glu Phe Ser Ala Asn Val MetAsn Thr 2160 2165 2170 2175 gag gct gac aag atc agc caa tct gag acc tacaga agg cgc cgt caa 6575 Glu Ala Asp Lys Ile Ser Gln Ser Glu Thr Tyr ArgArg Arg Arg Gln 2180 2185 2190 gag tgg gag atc caa agg aac aac gct gaggca gag ttg aag caa atc 6623 Glu Trp Glu Ile Gln Arg Asn Asn Ala Glu AlaGlu Leu Lys Gln Ile 2195 2200 2205 gat gct caa ctc aag tcc ttg gct gtcaga agg gag gct gct gtc ctc 6671 Asp Ala Gln Leu Lys Ser Leu Ala Val ArgArg Glu Ala Ala Val Leu 2210 2215 2220 cag aag acc tcc ctc aag acc caacag gag caa acc cag tcc cag ttg 6719 Gln Lys Thr Ser Leu Lys Thr Gln GlnGlu Gln Thr Gln Ser Gln Leu 2225 2230 2235 gct ttc ctc caa agg aag ttctcc aac cag gct ctc tac aac tgg ctc 6767 Ala Phe Leu Gln Arg Lys Phe SerAsn Gln Ala Leu Tyr Asn Trp Leu 2240 2245 2250 2255 aga ggc cgc ttg gctgcc atc tac ttc caa ttc tac gac ctt gct gtg 6815 Arg Gly Arg Leu Ala AlaIle Tyr Phe Gln Phe Tyr Asp Leu Ala Val 2260 2265 2270 gcc agg tgc ctcatg gct gag caa gcc tac cgc tgg gag ttg aac gat 6863 Ala Arg Cys Leu MetAla Glu Gln Ala Tyr Arg Trp Glu Leu Asn Asp 2275 2280 2285 gac tcc gccagg ttc atc aag cca ggt gct tgg caa ggc acc tac gct 6911 Asp Ser Ala ArgPhe Ile Lys Pro Gly Ala Trp Gln Gly Thr Tyr Ala 2290 2295 2300 ggt ctcctt gct ggt gag acc ctc atg ctc tcc ttg gct caa atg gag 6959 Gly Leu LeuAla Gly Glu Thr Leu Met Leu Ser Leu Ala Gln Met Glu 2305 2310 2315 gatgct cac ctc aag agg gac aag agg gct ttg gag gtg gag agg aca 7007 Asp AlaHis Leu Lys Arg Asp Lys Arg Ala Leu Glu Val Glu Arg Thr 2320 2325 23302335 gtc tcc ctt gct gag gtc tac gct ggt ctc cca aag gac aac ggt cca7055 Val Ser Leu Ala Glu Val Tyr Ala Gly Leu Pro Lys Asp Asn Gly Pro2340 2345 2350 ttc tcc ctt gct caa gag att gac aag ttg gtc agc caa ggttct ggt 7103 Phe Ser Leu Ala Gln Glu Ile Asp Lys Leu Val Ser Gln Gly SerGly 2355 2360 2365 tct gct ggt tct ggt aac aac aac ttg gct ttc ggc gctggt act gac 7151 Ser Ala Gly Ser Gly Asn Asn Asn Leu Ala Phe Gly Ala GlyThr Asp 2370 2375 2380 acc aag acc tcc ctc caa gcc tct gtc tcc ttc gctgac ctc aag atc 7199 Thr Lys Thr Ser Leu Gln Ala Ser Val Ser Phe Ala AspLeu Lys Ile 2385 2390 2395 agg gag gac tac cca gct tcc ctt ggc aag atcagg cgc atc aag caa 7247 Arg Glu Asp Tyr Pro Ala Ser Leu Gly Lys Ile ArgArg Ile Lys Gln 2400 2405 2410 2415 atc tct gtc acc ctc cca gct ctc ttgggt cca tac caa gat gtc caa 7295 Ile Ser Val Thr Leu Pro Ala Leu Leu GlyPro Tyr Gln Asp Val Gln 2420 2425 2430 gca atc ctc tcc tac ggt gac aaggct ggt ttg gcg aac ggt tgc gag 7343 Ala Ile Leu Ser Tyr Gly Asp Lys AlaGly Leu Ala Asn Gly Cys Glu 2435 2440 2445 gct ctt gct gtc tct cat ggcatg aac gac tct ggt caa ttc caa ctt 7391 Ala Leu Ala Val Ser His Gly MetAsn Asp Ser Gly Gln Phe Gln Leu 2450 2455 2460 gac ttc aac gat ggc aagttc ctc cca ttc gag ggc att gcc att gac 7439 Asp Phe Asn Asp Gly Lys PheLeu Pro Phe Glu Gly Ile Ala Ile Asp 2465 2470 2475 caa ggc acc ctc accctc tcc ttc cca aac gct tcc atg cca gag aag 7487 Gln Gly Thr Leu Thr LeuSer Phe Pro Asn Ala Ser Met Pro Glu Lys 2480 2485 2490 2495 gga aag caagcc acc atg ctc aag acc ctc aac gat atc atc ctc cac 7535 Gly Lys Gln AlaThr Met Leu Lys Thr Leu Asn Asp Ile Ile Leu His 2500 2505 2510 atc aggtac acc atc aag tgagctcgag aggcctgcgg ccgc 7577 Ile Arg Tyr Thr Ile Lys2515 4 7541 DNA Artificial Sequence CDS (3)..(7517) Description ofArtificial Sequencehemicot tcbA 4 cc atg gct cag aac tcc ctc agc tcc accatt gac acc atc tgc cag 47 Met Ala Gln Asn Ser Leu Ser Ser Thr Ile AspThr Ile Cys Gln 1 5 10 15 aag ctt caa ctc acc tgc cca gct gag atc gccctc tac cca ttc gac 95 Lys Leu Gln Leu Thr Cys Pro Ala Glu Ile Ala LeuTyr Pro Phe Asp 20 25 30 acc ttc cgt gag aag acc aga ggc atg gtc aac tggggt gag gcc aag 143 Thr Phe Arg Glu Lys Thr Arg Gly Met Val Asn Trp GlyGlu Ala Lys 35 40 45 agg atc tac gag att gct caa gct gag caa gac agg aacctc ctt cat 191 Arg Ile Tyr Glu Ile Ala Gln Ala Glu Gln Asp Arg Asn LeuLeu His 50 55 60 gag aag agg atc ttc gcc tac gct aac cca ttg ctc aag aacgct gtc 239 Glu Lys Arg Ile Phe Ala Tyr Ala Asn Pro Leu Leu Lys Asn AlaVal 65 70 75 agg ctt ggt acc agg caa atg ttg ggt ttc atc caa ggt tac tctgac 287 Arg Leu Gly Thr Arg Gln Met Leu Gly Phe Ile Gln Gly Tyr Ser Asp80 85 90 95 ttg ttc ggc aac agg gct gac aac tac gca gct cct ggt tct gttgct 335 Leu Phe Gly Asn Arg Ala Asp Asn Tyr Ala Ala Pro Gly Ser Val Ala100 105 110 agc atg ttc agc cca gct gcc tac ctc act gag ttg tac cgt gaggcc 383 Ser Met Phe Ser Pro Ala Ala Tyr Leu Thr Glu Leu Tyr Arg Glu Ala115 120 125 aag aac ctc cat gac agc tcc agc atc tac tac ctt gac aag aggcgc 431 Lys Asn Leu His Asp Ser Ser Ser Ile Tyr Tyr Leu Asp Lys Arg Arg130 135 140 cca gac ctt gct tcc ttg atg ctc tcc cag aag aac atg gat gaggag 479 Pro Asp Leu Ala Ser Leu Met Leu Ser Gln Lys Asn Met Asp Glu Glu145 150 155 atc agc acc ttg gct ctc tcc aac gag ctt tgc ttg gct ggc attgag 527 Ile Ser Thr Leu Ala Leu Ser Asn Glu Leu Cys Leu Ala Gly Ile Glu160 165 170 175 acc aag act ggc aag tcc caa gat gag gtc atg gac atg ctctcc acc 575 Thr Lys Thr Gly Lys Ser Gln Asp Glu Val Met Asp Met Leu SerThr 180 185 190 tac cgc ctc tct ggt gag act cca tac cac cat gct tac gagact gtc 623 Tyr Arg Leu Ser Gly Glu Thr Pro Tyr His His Ala Tyr Glu ThrVal 195 200 205 agg gag att gtc cat gag agg gac cca ggt ttc cgc cac ctctcc caa 671 Arg Glu Ile Val His Glu Arg Asp Pro Gly Phe Arg His Leu SerGln 210 215 220 gct ccc att gtg gct gcc aag ttg gac cca gtc acc ctc ttgggc atc 719 Ala Pro Ile Val Ala Ala Lys Leu Asp Pro Val Thr Leu Leu GlyIle 225 230 235 tcc agc cac atc agc cca gag ttg tac aac ctt ctc att gaggag atc 767 Ser Ser His Ile Ser Pro Glu Leu Tyr Asn Leu Leu Ile Glu GluIle 240 245 250 255 cca gag aag gat gag gca gct ttg gac acc ctc tac aagacc aac ttc 815 Pro Glu Lys Asp Glu Ala Ala Leu Asp Thr Leu Tyr Lys ThrAsn Phe 260 265 270 ggt gac atc acc act gct caa ctc atg agc cca tcc tacttg gcc agg 863 Gly Asp Ile Thr Thr Ala Gln Leu Met Ser Pro Ser Tyr LeuAla Arg 275 280 285 tac tac ggt gtc tct cca gag gac att gct tac gtc accaca agc ctc 911 Tyr Tyr Gly Val Ser Pro Glu Asp Ile Ala Tyr Val Thr ThrSer Leu 290 295 300 tcc cat gtg ggt tac tcc tct gac atc ctt gtc atc ccactc gtg gat 959 Ser His Val Gly Tyr Ser Ser Asp Ile Leu Val Ile Pro LeuVal Asp 305 310 315 ggt gtg ggc aag atg gag gtt gtc agg gtc acc agg actcca tct gac 1007 Gly Val Gly Lys Met Glu Val Val Arg Val Thr Arg Thr ProSer Asp 320 325 330 335 aac tac acc tcc cag acc aac tac att gag ttg taccca caa ggt ggt 1055 Asn Tyr Thr Ser Gln Thr Asn Tyr Ile Glu Leu Tyr ProGln Gly Gly 340 345 350 gac aac tac ctc atc aag tac aac ctc tcc aac tctttc ggt ttg gat 1103 Asp Asn Tyr Leu Ile Lys Tyr Asn Leu Ser Asn Ser PheGly Leu Asp 355 360 365 gac ttc tac ctc cag tac aag gat ggt tct gct gactgg act gag att 1151 Asp Phe Tyr Leu Gln Tyr Lys Asp Gly Ser Ala Asp TrpThr Glu Ile 370 375 380 gct cac aac cca tac cca gac atg gtc atc aac cagaag tac gag tcc 1199 Ala His Asn Pro Tyr Pro Asp Met Val Ile Asn Gln LysTyr Glu Ser 385 390 395 caa gcc acc atc aag aga tct gac tct gac aac atcctc tcc att ggt 1247 Gln Ala Thr Ile Lys Arg Ser Asp Ser Asp Asn Ile LeuSer Ile Gly 400 405 410 415 ctc caa agg tgg cac tct ggt tcc tac aac ttcgct gct gcc aac ttc 1295 Leu Gln Arg Trp His Ser Gly Ser Tyr Asn Phe AlaAla Ala Asn Phe 420 425 430 aag att gac caa tac tct cca aag gct ttc ctcttg aag atg aac aag 1343 Lys Ile Asp Gln Tyr Ser Pro Lys Ala Phe Leu LeuLys Met Asn Lys 435 440 445 gcc atc agg ctc ttg aag gcc act ggt ctc tccttc gcc acc ctt gag 1391 Ala Ile Arg Leu Leu Lys Ala Thr Gly Leu Ser PheAla Thr Leu Glu 450 455 460 agg att gtg gac tct gtc aac tcc acc aag tccatc act gtg gag gtc 1439 Arg Ile Val Asp Ser Val Asn Ser Thr Lys Ser IleThr Val Glu Val 465 470 475 ctc aac aag gtc tac aga gtc aag ttc tac attgac cgc tac ggc atc 1487 Leu Asn Lys Val Tyr Arg Val Lys Phe Tyr Ile AspArg Tyr Gly Ile 480 485 490 495 tct gag gag act gct gcc atc ctt gcc aacatc aac atc tcc cag caa 1535 Ser Glu Glu Thr Ala Ala Ile Leu Ala Asn IleAsn Ile Ser Gln Gln 500 505 510 gct gtc ggc aac cag ctc tcc caa ttc gagcaa ctc ttc aac cac cct 1583 Ala Val Gly Asn Gln Leu Ser Gln Phe Glu GlnLeu Phe Asn His Pro 515 520 525 cca ctc aac ggc atc cgc tac gag atc agcgag gac aac tcc aag cac 1631 Pro Leu Asn Gly Ile Arg Tyr Glu Ile Ser GluAsp Asn Ser Lys His 530 535 540 ctc cca aac cca gac ctc aac ctc aag ccagac tcc act ggt gat gac 1679 Leu Pro Asn Pro Asp Leu Asn Leu Lys Pro AspSer Thr Gly Asp Asp 545 550 555 caa agg aag gct gtc ctc aag agg gct ttccaa gtc aac gct tct gag 1727 Gln Arg Lys Ala Val Leu Lys Arg Ala Phe GlnVal Asn Ala Ser Glu 560 565 570 575 ctt tac caa atg ctc ttg atc act gacagg aag gag gat ggt gtc atc 1775 Leu Tyr Gln Met Leu Leu Ile Thr Asp ArgLys Glu Asp Gly Val Ile 580 585 590 aag aac aac ttg gag aac ctc tct gacctc tac ctt gtc tcc ctc ttg 1823 Lys Asn Asn Leu Glu Asn Leu Ser Asp LeuTyr Leu Val Ser Leu Leu 595 600 605 gcc caa atc cac aac ttg acc att gctgag ttg aac atc ctc ttg gtc 1871 Ala Gln Ile His Asn Leu Thr Ile Ala GluLeu Asn Ile Leu Leu Val 610 615 620 atc tgc ggt tac ggt gac acc aac atctac caa atc act gac gac aac 1919 Ile Cys Gly Tyr Gly Asp Thr Asn Ile TyrGln Ile Thr Asp Asp Asn 625 630 635 ctt gcc aag att gtg gag acc ctc ttgtgg atc acc caa tgg ctc aag 1967 Leu Ala Lys Ile Val Glu Thr Leu Leu TrpIle Thr Gln Trp Leu Lys 640 645 650 655 acc cag aag tgg act gtc aca gacctc ttc ctc atg acc act gcc acc 2015 Thr Gln Lys Trp Thr Val Thr Asp LeuPhe Leu Met Thr Thr Ala Thr 660 665 670 tac tcc acc act ctc act cca gagatt tcc aac ctc act gcc acc ctc 2063 Tyr Ser Thr Thr Leu Thr Pro Glu IleSer Asn Leu Thr Ala Thr Leu 675 680 685 agc tcc acc ctc cac ggc aag gagtcc ctc att ggt gag gac ctc aag 2111 Ser Ser Thr Leu His Gly Lys Glu SerLeu Ile Gly Glu Asp Leu Lys 690 695 700 agg gca atg gct cca tgc ttc acctct gct ctc cac ctc acc tcc caa 2159 Arg Ala Met Ala Pro Cys Phe Thr SerAla Leu His Leu Thr Ser Gln 705 710 715 gag gtg gct tac gac ctc ctt ctctgg att gac caa atc caa cca gct 2207 Glu Val Ala Tyr Asp Leu Leu Leu TrpIle Asp Gln Ile Gln Pro Ala 720 725 730 735 caa atc act gtg gat ggt ttctgg gag gag gtc caa acc act cca acc 2255 Gln Ile Thr Val Asp Gly Phe TrpGlu Glu Val Gln Thr Thr Pro Thr 740 745 750 tcc ctc aag gtc atc acc ttcgct caa gtc ttg gct caa ctc tcc ctc 2303 Ser Leu Lys Val Ile Thr Phe AlaGln Val Leu Ala Gln Leu Ser Leu 755 760 765 atc tac aga agg att ggt ctctct gag act gag ttg tcc ctc att gtc 2351 Ile Tyr Arg Arg Ile Gly Leu SerGlu Thr Glu Leu Ser Leu Ile Val 770 775 780 acc caa tcc agc ctc ttg gtcgct ggc aag tcc atc ctt gat cat ggt 2399 Thr Gln Ser Ser Leu Leu Val AlaGly Lys Ser Ile Leu Asp His Gly 785 790 795 ctc ttg acc ctc atg gct cttgag ggt ttc cac acc tgg gtc aac ggt 2447 Leu Leu Thr Leu Met Ala Leu GluGly Phe His Thr Trp Val Asn Gly 800 805 810 815 ttg ggt caa cat gct tccctc atc ttg gct gca ctc aag gat ggt gct 2495 Leu Gly Gln His Ala Ser LeuIle Leu Ala Ala Leu Lys Asp Gly Ala 820 825 830 ctc acc gtc acc gat gtggct caa gcc atg aac aag gag gag tcc ctc 2543 Leu Thr Val Thr Asp Val AlaGln Ala Met Asn Lys Glu Glu Ser Leu 835 840 845 ttg caa atg gct gcc aaccag gtg gag aag gac ctc acc aag ctc acc 2591 Leu Gln Met Ala Ala Asn GlnVal Glu Lys Asp Leu Thr Lys Leu Thr 850 855 860 tcc tgg acc caa atc gatgcc atc ctc caa tgg ctc caa atg tcc tct 2639 Ser Trp Thr Gln Ile Asp AlaIle Leu Gln Trp Leu Gln Met Ser Ser 865 870 875 gct ctt gct gtc agc ccattg gac ctt gct ggc atg atg gct ctc aag 2687 Ala Leu Ala Val Ser Pro LeuAsp Leu Ala Gly Met Met Ala Leu Lys 880 885 890 895 tac ggc att gat cacaac tac gct gcc tgg caa gca gct gcc gct gcc 2735 Tyr Gly Ile Asp His AsnTyr Ala Ala Trp Gln Ala Ala Ala Ala Ala 900 905 910 ctc atg gct gac catgcc aac cag gct cag aag aag ttg gat gag acc 2783 Leu Met Ala Asp His AlaAsn Gln Ala Gln Lys Lys Leu Asp Glu Thr 915 920 925 ttc tcc aag gct ctctgc aac tac tac atc aac gcc gtg gtt gac tct 2831 Phe Ser Lys Ala Leu CysAsn Tyr Tyr Ile Asn Ala Val Val Asp Ser 930 935 940 gct gcc ggt gtc agggac agg aac ggt ctc tac acc tac ctc ttg att 2879 Ala Ala Gly Val Arg AspArg Asn Gly Leu Tyr Thr Tyr Leu Leu Ile 945 950 955 gac aac cag gtc tctgct gat gtc atc acc tcc aga att gct gag gcc 2927 Asp Asn Gln Val Ser AlaAsp Val Ile Thr Ser Arg Ile Ala Glu Ala 960 965 970 975 att gct ggc atccaa ctc tac gtc aac agg gct ctc aac agg gat gag 2975 Ile Ala Gly Ile GlnLeu Tyr Val Asn Arg Ala Leu Asn Arg Asp Glu 980 985 990 ggt cag ttg gcttct gat gtc tcc acc agg caa ttc ttc acc gac tgg 3023 Gly Gln Leu Ala SerAsp Val Ser Thr Arg Gln Phe Phe Thr Asp Trp 995 1000 1005 gag agg tacaac aag agg tac tcc acc tgg gct ggt gtc tct gag ttg 3071 Glu Arg Tyr AsnLys Arg Tyr Ser Thr Trp Ala Gly Val Ser Glu Leu 1010 1015 1020 gtc tactac cca gag aac tac gtg gac cca acc caa agg att ggt cag 3119 Val Tyr TyrPro Glu Asn Tyr Val Asp Pro Thr Gln Arg Ile Gly Gln 1025 1030 1035 accaag atg atg gat gct ttg ctc caa tcc atc aac cag tcc caa ctc 3167 Thr LysMet Met Asp Ala Leu Leu Gln Ser Ile Asn Gln Ser Gln Leu 1040 1045 10501055 aac gct gac act gtg gag gat gct ttc aag acc tac ctc acc tcc ttc3215 Asn Ala Asp Thr Val Glu Asp Ala Phe Lys Thr Tyr Leu Thr Ser Phe1060 1065 1070 gag caa gtg gcc aac ctc aag gtc atc tct gct tac cat gacaac gtc 3263 Glu Gln Val Ala Asn Leu Lys Val Ile Ser Ala Tyr His Asp AsnVal 1075 1080 1085 aac gtg gac caa ggt ctc acc tac ttc att ggc att gaccaa gcc gct 3311 Asn Val Asp Gln Gly Leu Thr Tyr Phe Ile Gly Ile Asp GlnAla Ala 1090 1095 1100 cct ggc acc tac tac tgg agg tct gtg gac cac tccaag tgc gag aac 3359 Pro Gly Thr Tyr Tyr Trp Arg Ser Val Asp His Ser LysCys Glu Asn 1105 1110 1115 ggc aag ttc gct gcc aac gct tgg ggt gag tggaac aag atc acc tgc 3407 Gly Lys Phe Ala Ala Asn Ala Trp Gly Glu Trp AsnLys Ile Thr Cys 1120 1125 1130 1135 gct gtc aac cct tgg aag aac atc atcagg cca gtg gtc tac atg tcc 3455 Ala Val Asn Pro Trp Lys Asn Ile Ile ArgPro Val Val Tyr Met Ser 1140 1145 1150 aga ctc tac ttg ctc tgg ctt gagcaa cag tcc aag aag tct gat gac 3503 Arg Leu Tyr Leu Leu Trp Leu Glu GlnGln Ser Lys Lys Ser Asp Asp 1155 1160 1165 ggc aag aca act atc tac cagtac aac ctc aag ttg gct cac atc cgc 3551 Gly Lys Thr Thr Ile Tyr Gln TyrAsn Leu Lys Leu Ala His Ile Arg 1170 1175 1180 tac gat ggt tcc tgg aacact cca ttc acc ttc gat gtc act gag aag 3599 Tyr Asp Gly Ser Trp Asn ThrPro Phe Thr Phe Asp Val Thr Glu Lys 1185 1190 1195 gtc aag aac tac acctcc agc act gat gca gct gag tcc ctt ggt ctc 3647 Val Lys Asn Tyr Thr SerSer Thr Asp Ala Ala Glu Ser Leu Gly Leu 1200 1205 1210 1215 tac tgc actggt tac caa ggt gag gac acc ctc ttg gtc atg ttc tac 3695 Tyr Cys Thr GlyTyr Gln Gly Glu Asp Thr Leu Leu Val Met Phe Tyr 1220 1225 1230 tcc atgcaa tcc agc tac tcc agc tac act gac aac aac gct cca gtc 3743 Ser Met GlnSer Ser Tyr Ser Ser Tyr Thr Asp Asn Asn Ala Pro Val 1235 1240 1245 actggt ctc tac atc ttc gct gac atg tcc tct gac aac atg acc aac 3791 Thr GlyLeu Tyr Ile Phe Ala Asp Met Ser Ser Asp Asn Met Thr Asn 1250 1255 1260gct caa gcc acc aac tac tgg aac aac tcc tac cca caa ttc gac act 3839 AlaGln Ala Thr Asn Tyr Trp Asn Asn Ser Tyr Pro Gln Phe Asp Thr 1265 12701275 gtc atg gct gac cca gac tct gac aac aag aag gtc atc acc agg cgt3887 Val Met Ala Asp Pro Asp Ser Asp Asn Lys Lys Val Ile Thr Arg Arg1280 1285 1290 1295 gtc aac aac cgc tac gct gag gac tac gag atc cca agctct gtc acc 3935 Val Asn Asn Arg Tyr Ala Glu Asp Tyr Glu Ile Pro Ser SerVal Thr 1300 1305 1310 tcc aac agc aac tac tcc tgg ggt gac cac tcc ctcacc atg ctc tac 3983 Ser Asn Ser Asn Tyr Ser Trp Gly Asp His Ser Leu ThrMet Leu Tyr 1315 1320 1325 ggt ggc tct gtc cca aac atc acc ttc gag tctgca gct gag gac ctc 4031 Gly Gly Ser Val Pro Asn Ile Thr Phe Glu Ser AlaAla Glu Asp Leu 1330 1335 1340 agg ctc tcc acc aac atg gct ctc tcc atcatt cac aac ggt tac gct 4079 Arg Leu Ser Thr Asn Met Ala Leu Ser Ile IleHis Asn Gly Tyr Ala 1345 1350 1355 ggc acc agg cgc atc caa tgc aac ctcatg aag caa tac gct tcc ctt 4127 Gly Thr Arg Arg Ile Gln Cys Asn Leu MetLys Gln Tyr Ala Ser Leu 1360 1365 1370 1375 ggt gac aag ttc att atc tacgac tcc agc ttc gat gac gcc aac agg 4175 Gly Asp Lys Phe Ile Ile Tyr AspSer Ser Phe Asp Asp Ala Asn Arg 1380 1385 1390 ttc aac ttg gtc cca ctcttc aag ttc ggc aag gat gag aac tct gat 4223 Phe Asn Leu Val Pro Leu PheLys Phe Gly Lys Asp Glu Asn Ser Asp 1395 1400 1405 gac tcc atc tgc atctac aac gag aac cca agc tct gag gac aag aag 4271 Asp Ser Ile Cys Ile TyrAsn Glu Asn Pro Ser Ser Glu Asp Lys Lys 1410 1415 1420 tgg tac ttc agctcc aag gac gac aac aag act gct gac tac aac ggt 4319 Trp Tyr Phe Ser SerLys Asp Asp Asn Lys Thr Ala Asp Tyr Asn Gly 1425 1430 1435 ggc acc caatgc att gat gct ggc acc tcc aac aag gac ttc tac tac 4367 Gly Thr Gln CysIle Asp Ala Gly Thr Ser Asn Lys Asp Phe Tyr Tyr 1440 1445 1450 1455 aacctc caa gag att gag gtc atc tct gtc act ggt ggc tac tgg tcc 4415 Asn LeuGln Glu Ile Glu Val Ile Ser Val Thr Gly Gly Tyr Trp Ser 1460 1465 1470agc tac aag atc agc aac ccc atc aac atc aac act ggc att gac tct 4463 SerTyr Lys Ile Ser Asn Pro Ile Asn Ile Asn Thr Gly Ile Asp Ser 1475 14801485 gcc aag gtc aag gtc act gtc aag gct ggt ggc gat gac caa atc ttc4511 Ala Lys Val Lys Val Thr Val Lys Ala Gly Gly Asp Asp Gln Ile Phe1490 1495 1500 act gct gac aac tcc acc tac gtc cca cag caa cct gct ccatcc ttc 4559 Thr Ala Asp Asn Ser Thr Tyr Val Pro Gln Gln Pro Ala Pro SerPhe 1505 1510 1515 gag gag atg atc tac caa ttc aac aac ctc acc att gactgc aag aac 4607 Glu Glu Met Ile Tyr Gln Phe Asn Asn Leu Thr Ile Asp CysLys Asn 1520 1525 1530 1535 ctc aac ttc att gac aac cag gct cac att gagatt gac ttc act gcc 4655 Leu Asn Phe Ile Asp Asn Gln Ala His Ile Glu IleAsp Phe Thr Ala 1540 1545 1550 aca gct caa gat ggc cgc ttc ttg ggt gctgag acc ttc atc att cca 4703 Thr Ala Gln Asp Gly Arg Phe Leu Gly Ala GluThr Phe Ile Ile Pro 1555 1560 1565 gtc acc aag aag gtc ctt ggc act gagaac gtc att gct ctc tac tct 4751 Val Thr Lys Lys Val Leu Gly Thr Glu AsnVal Ile Ala Leu Tyr Ser 1570 1575 1580 gag aac aac ggt gtc cag tac atgcaa att ggt gct tac aga acc agg 4799 Glu Asn Asn Gly Val Gln Tyr Met GlnIle Gly Ala Tyr Arg Thr Arg 1585 1590 1595 ctc aac acc ctc ttc gct caacag ttg gtc tcc cgt gcc aac aga ggc 4847 Leu Asn Thr Leu Phe Ala Gln GlnLeu Val Ser Arg Ala Asn Arg Gly 1600 1605 1610 1615 att gat gct gtc ctcagc atg gag act cag aac atc caa gag cca caa 4895 Ile Asp Ala Val Leu SerMet Glu Thr Gln Asn Ile Gln Glu Pro Gln 1620 1625 1630 ctt ggt gct ggcacc tac gtc caa ctt gtc ttg gac aag tac gat gag 4943 Leu Gly Ala Gly ThrTyr Val Gln Leu Val Leu Asp Lys Tyr Asp Glu 1635 1640 1645 tcc att catggc acc aac aag tcc ttc gcc att gag tac gtg gac atc 4991 Ser Ile His GlyThr Asn Lys Ser Phe Ala Ile Glu Tyr Val Asp Ile 1650 1655 1660 ttc aaggag aac gac tcc ttc gtc atc tac caa ggt gag ttg tct gag 5039 Phe Lys GluAsn Asp Ser Phe Val Ile Tyr Gln Gly Glu Leu Ser Glu 1665 1670 1675 acctcc caa act gtg gtc aag gtc ttc ctc tcc tac ttc att gag gcc 5087 Thr SerGln Thr Val Val Lys Val Phe Leu Ser Tyr Phe Ile Glu Ala 1680 1685 16901695 acc ggt aac aag aac cac ctc tgg gtc agg gcc aag tac cag aag gag5135 Thr Gly Asn Lys Asn His Leu Trp Val Arg Ala Lys Tyr Gln Lys Glu1700 1705 1710 acc act gac aag atc ctc ttc gac agg act gat gag aag gaccca cat 5183 Thr Thr Asp Lys Ile Leu Phe Asp Arg Thr Asp Glu Lys Asp ProHis 1715 1720 1725 ggt tgg ttc ctc tct gat gac cac aag acc ttc tct ggtctc agc tct 5231 Gly Trp Phe Leu Ser Asp Asp His Lys Thr Phe Ser Gly LeuSer Ser 1730 1735 1740 gct caa gct ctc aag aac gac tct gag cca atg gacttc tct ggt gcc 5279 Ala Gln Ala Leu Lys Asn Asp Ser Glu Pro Met Asp PheSer Gly Ala 1745 1750 1755 aac gct ctc tac ttc tgg gag ttg ttc tac tacact cca atg atg atg 5327 Asn Ala Leu Tyr Phe Trp Glu Leu Phe Tyr Tyr ThrPro Met Met Met 1760 1765 1770 1775 gct cac agg ctc ctt caa gag cag aacttc gat gct gcc aac cac tgg 5375 Ala His Arg Leu Leu Gln Glu Gln Asn PheAsp Ala Ala Asn His Trp 1780 1785 1790 ttc cgc tac gtc tgg agc cca tctggt tac att gtg gat ggc aag att 5423 Phe Arg Tyr Val Trp Ser Pro Ser GlyTyr Ile Val Asp Gly Lys Ile 1795 1800 1805 gcc atc tac cac tgg aac gtcagg cca ttg gag gag gac acc tcc tgg 5471 Ala Ile Tyr His Trp Asn Val ArgPro Leu Glu Glu Asp Thr Ser Trp 1810 1815 1820 aac gct cag caa ctt gactcc act gac cca gat gct gtg gct caa gat 5519 Asn Ala Gln Gln Leu Asp SerThr Asp Pro Asp Ala Val Ala Gln Asp 1825 1830 1835 gac cca atg cac tacaag gtg gcc acc ttc atg gcc acc ttg gac ctt 5567 Asp Pro Met His Tyr LysVal Ala Thr Phe Met Ala Thr Leu Asp Leu 1840 1845 1850 1855 ctc atg gccaga ggt gat gct gcc tac cgc caa ttg gag agg gac acc 5615 Leu Met Ala ArgGly Asp Ala Ala Tyr Arg Gln Leu Glu Arg Asp Thr 1860 1865 1870 ttg gctgag gcc aag atg tgg tac acc caa gct ctc aac ttg ctg ggt 5663 Leu Ala GluAla Lys Met Trp Tyr Thr Gln Ala Leu Asn Leu Leu Gly 1875 1880 1885 gatgag cca caa gtc atg ctc tcc aca acc tgg gcc aac cca acc ttg 5711 Asp GluPro Gln Val Met Leu Ser Thr Thr Trp Ala Asn Pro Thr Leu 1890 1895 1900ggc aac gct gcc tcc aag acc aca caa cag gtc agg caa cag gtc ctc 5759 GlyAsn Ala Ala Ser Lys Thr Thr Gln Gln Val Arg Gln Gln Val Leu 1905 19101915 acc caa ctc agg ctc aac tct aga gtc aag act cca ctc ttg ggc act5807 Thr Gln Leu Arg Leu Asn Ser Arg Val Lys Thr Pro Leu Leu Gly Thr1920 1925 1930 1935 gcc aac tcc ctc act gct ctc ttc ctc cca caa gag aactcc aaa ctt 5855 Ala Asn Ser Leu Thr Ala Leu Phe Leu Pro Gln Glu Asn SerLys Leu 1940 1945 1950 aag ggt tac tgg agg acc ctt gct caa cgc atg ttcaac ctc agg cac 5903 Lys Gly Tyr Trp Arg Thr Leu Ala Gln Arg Met Phe AsnLeu Arg His 1955 1960 1965 aac ctc tcc att gat ggt caa cca ctc tcc ttgcca ctc tac gct aag 5951 Asn Leu Ser Ile Asp Gly Gln Pro Leu Ser Leu ProLeu Tyr Ala Lys 1970 1975 1980 cca gct gac cca aag gct ctc ctt tcc gctgct gtc tcc gca tcc caa 5999 Pro Ala Asp Pro Lys Ala Leu Leu Ser Ala AlaVal Ser Ala Ser Gln 1985 1990 1995 ggt ggt gct gac ctc cca aag gct ccactc acc atc cac agg ttc cca 6047 Gly Gly Ala Asp Leu Pro Lys Ala Pro LeuThr Ile His Arg Phe Pro 2000 2005 2010 2015 caa atg ttg gag ggt gcc cgtggt ctt gtc aac cag ctc atc caa ttc 6095 Gln Met Leu Glu Gly Ala Arg GlyLeu Val Asn Gln Leu Ile Gln Phe 2020 2025 2030 ggt tcc tct ctc ctt ggttac tct gag agg caa gat gct gag gcc atg 6143 Gly Ser Ser Leu Leu Gly TyrSer Glu Arg Gln Asp Ala Glu Ala Met 2035 2040 2045 tcc caa ctc ttg caaacc cag gct tct gag ttg atc ctc acc tcc atc 6191 Ser Gln Leu Leu Gln ThrGln Ala Ser Glu Leu Ile Leu Thr Ser Ile 2050 2055 2060 agg atg caa gacaac cag ctt gct gag ttg gac tct gag aag act gct 6239 Arg Met Gln Asp AsnGln Leu Ala Glu Leu Asp Ser Glu Lys Thr Ala 2065 2070 2075 ctc caa gtctcc ctt gct ggt gtc caa cag agg ttc gac agc tac tcc 6287 Leu Gln Val SerLeu Ala Gly Val Gln Gln Arg Phe Asp Ser Tyr Ser 2080 2085 2090 2095 caactc tac gag gag aac atc aac gct ggt gag caa agg gct ttg gct 6335 Gln LeuTyr Glu Glu Asn Ile Asn Ala Gly Glu Gln Arg Ala Leu Ala 2100 2105 2110ctc agg tct gag tct gcc att gag tcc caa ggt gct caa atc tcc cgc 6383 LeuArg Ser Glu Ser Ala Ile Glu Ser Gln Gly Ala Gln Ile Ser Arg 2115 21202125 atg gct ggt gct ggc gtg gac atg gct cca aac atc ttc ggt ctt gct6431 Met Ala Gly Ala Gly Val Asp Met Ala Pro Asn Ile Phe Gly Leu Ala2130 2135 2140 gat ggt ggc atg cac tac ggt gcc att gct tac gcc att gctgat ggc 6479 Asp Gly Gly Met His Tyr Gly Ala Ile Ala Tyr Ala Ile Ala AspGly 2145 2150 2155 att gag ctt tct gct tct gcc aag atg gtt gat gct gagaag gtg gct 6527 Ile Glu Leu Ser Ala Ser Ala Lys Met Val Asp Ala Glu LysVal Ala 2160 2165 2170 2175 caa tct gaa atc tac cgt cgc aga cgc caa gaatgg aag atc caa agg 6575 Gln Ser Glu Ile Tyr Arg Arg Arg Arg Gln Glu TrpLys Ile Gln Arg 2180 2185 2190 gac aac gct caa gct gag atc aac cag ctcaac gct caa ctt gag tcc 6623 Asp Asn Ala Gln Ala Glu Ile Asn Gln Leu AsnAla Gln Leu Glu Ser 2195 2200 2205 ctc agc atc agg cgt gag gct gct gagatg cag aag gag tac ctc aag 6671 Leu Ser Ile Arg Arg Glu Ala Ala Glu MetGln Lys Glu Tyr Leu Lys 2210 2215 2220 acc caa cag gct caa gct cag gctcaa ctc acc ttc ctc agg tcc aag 6719 Thr Gln Gln Ala Gln Ala Gln Ala GlnLeu Thr Phe Leu Arg Ser Lys 2225 2230 2235 ttc tcc aac cag gct ctc tactcc tgg ctc aga ggc cgc ctc tct ggc 6767 Phe Ser Asn Gln Ala Leu Tyr SerTrp Leu Arg Gly Arg Leu Ser Gly 2240 2245 2250 2255 atc tac ttc caa ttctac gac ttg gct gtc tcc cgc tgc ctc atg gct 6815 Ile Tyr Phe Gln Phe TyrAsp Leu Ala Val Ser Arg Cys Leu Met Ala 2260 2265 2270 gag caa tcc taccaa tgg gag gcc aac gac aac agc atc tcc ttc gtc 6863 Glu Gln Ser Tyr GlnTrp Glu Ala Asn Asp Asn Ser Ile Ser Phe Val 2275 2280 2285 aag cca ggtgct tgg caa ggc acc tac gct ggt ctc ctt tgc ggt gag 6911 Lys Pro Gly AlaTrp Gln Gly Thr Tyr Ala Gly Leu Leu Cys Gly Glu 2290 2295 2300 gct ctcatc cag aac ttg gct caa atg gag gag gct tac ctc aag tgg 6959 Ala Leu IleGln Asn Leu Ala Gln Met Glu Glu Ala Tyr Leu Lys Trp 2305 2310 2315 gagtcc aga gct ttg gag gta gag agg act gtc tcc ctt gct gta gtc 7007 Glu SerArg Ala Leu Glu Val Glu Arg Thr Val Ser Leu Ala Val Val 2320 2325 23302335 tac gac tcc ttg gag ggc aac gac agg ttc aac ctt gct gag caa atc7055 Tyr Asp Ser Leu Glu Gly Asn Asp Arg Phe Asn Leu Ala Glu Gln Ile2340 2345 2350 cca gct ctc ttg gac aag ggt gag ggc act gct ggc acc aaggag aac 7103 Pro Ala Leu Leu Asp Lys Gly Glu Gly Thr Ala Gly Thr Lys GluAsn 2355 2360 2365 ggt ctc tcc ttg gcc aac gcc atc ctc tct gct tct gtcaag ctc tct 7151 Gly Leu Ser Leu Ala Asn Ala Ile Leu Ser Ala Ser Val LysLeu Ser 2370 2375 2380 gac ctc aag ttg ggt act gac tac cca gac tcc attgtg ggt tcc aac 7199 Asp Leu Lys Leu Gly Thr Asp Tyr Pro Asp Ser Ile ValGly Ser Asn 2385 2390 2395 aag gtc aga agg atc aag caa atc tct gtc tccctc cca gct ttg gtg 7247 Lys Val Arg Arg Ile Lys Gln Ile Ser Val Ser LeuPro Ala Leu Val 2400 2405 2410 2415 ggt cca tac caa gat gtc caa gcc atgctc tcc tac ggt ggc tcc acc 7295 Gly Pro Tyr Gln Asp Val Gln Ala Met LeuSer Tyr Gly Gly Ser Thr 2420 2425 2430 caa ctc cca aag ggt tgc tct gctttg gct gtc tcc cac ggc acc aac 7343 Gln Leu Pro Lys Gly Cys Ser Ala LeuAla Val Ser His Gly Thr Asn 2435 2440 2445 gac tct ggt caa ttc caa cttgac ttc aac gat ggc aag tac ctc cca 7391 Asp Ser Gly Gln Phe Gln Leu AspPhe Asn Asp Gly Lys Tyr Leu Pro 2450 2455 2460 ttc gaa ggc att gct ttggat gac caa ggc acc ctc aac ctc caa ttc 7439 Phe Glu Gly Ile Ala Leu AspAsp Gln Gly Thr Leu Asn Leu Gln Phe 2465 2470 2475 cca aac gcc act gacaag cag aag gcc atc ctc caa acc atg tct gac 7487 Pro Asn Ala Thr Asp LysGln Lys Ala Ile Leu Gln Thr Met Ser Asp 2480 2485 2490 2495 atc atc ctccac atc agg tac acc atc agg tgagctcgag aggcctgcgg 7537 Ile Ile Leu HisIle Arg Tyr Thr Ile Arg 2500 2505 5 63 DNA Artificial SequenceDescription of Artificial Sequencehemicot sequence encoding ER signalfrom 15 kDa zein from Black Mexican Sweet maize 5 atg gct aag atg gtcatt gtg ctt gtg gtc tgc ttg gct ctc tct gct 48 Met Ala Lys Met Val IleVal Leu Val Val Cys Leu Ala Leu Ser Ala 1 5 10 15 gcc tgt gct tca gcc 63Ala Cys Ala Ser Ala 20 6 7621 DNA Artificial Sequence Description ofArtificial Sequencehemicot tcdA fused to the modified 15 kDa zeinendoplasmic reticulum signal peptide 6 ncc atg gct aag atg gtc att gtgctt gtg gtc tgc ttg gct ctc tct 48 Met Ala Lys Met Val Ile Val Leu ValVal Cys Leu Ala Leu Ser 1 5 10 15 gct gcc tgt gct tca gcc atg aac gagtcc gtc aag gag atc cca gac 96 Ala Ala Cys Ala Ser Ala Met Asn Glu SerVal Lys Glu Ile Pro Asp 20 25 30 gtc ctc aag tcc caa tgc ggt ttc aac tgcctc act gac atc tcc cac 144 Val Leu Lys Ser Gln Cys Gly Phe Asn Cys LeuThr Asp Ile Ser His 35 40 45 agc tcc ttc aac gag ttc aga caa caa gtc tctgag cac ctc tcc tgg 192 Ser Ser Phe Asn Glu Phe Arg Gln Gln Val Ser GluHis Leu Ser Trp 50 55 60 tcc gag acc cat gac ctc tac cat gac gct cag caagct cag aag gac 240 Ser Glu Thr His Asp Leu Tyr His Asp Ala Gln Gln AlaGln Lys Asp 65 70 75 aac agg ctc tac gag gct agg atc ctc aag agg gct aaccca caa ctc 288 Asn Arg Leu Tyr Glu Ala Arg Ile Leu Lys Arg Ala Asn ProGln Leu 80 85 90 95 cag aac gct gtc cac ctc gcc atc ttg gct cca aac gctgag ttg att 336 Gln Asn Ala Val His Leu Ala Ile Leu Ala Pro Asn Ala GluLeu Ile 100 105 110 ggt tac aac aac cag ttc tct ggc aga gct agc cag tacgtg gct cct 384 Gly Tyr Asn Asn Gln Phe Ser Gly Arg Ala Ser Gln Tyr ValAla Pro 115 120 125 ggt aca gtc tcc tcc atg ttc agc cca gcc gct tac ctcact gag ttg 432 Gly Thr Val Ser Ser Met Phe Ser Pro Ala Ala Tyr Leu ThrGlu Leu 130 135 140 tac cgc gag gct agg aac ctt cat gct tct gac tcc gtctac tac ttg 480 Tyr Arg Glu Ala Arg Asn Leu His Ala Ser Asp Ser Val TyrTyr Leu 145 150 155 gac aca cgc aga cca gac ctc aag agc atg gcc ctc agccaa cag aac 528 Asp Thr Arg Arg Pro Asp Leu Lys Ser Met Ala Leu Ser GlnGln Asn 160 165 170 175 atg gac att gag ttg tcc acc ctc tcc ttg agc aacgag ctt ctc ttg 576 Met Asp Ile Glu Leu Ser Thr Leu Ser Leu Ser Asn GluLeu Leu Leu 180 185 190 gag tcc atc aag act gag agc aag ttg gag aac tacacc aag gtc atg 624 Glu Ser Ile Lys Thr Glu Ser Lys Leu Glu Asn Tyr ThrLys Val Met 195 200 205 gag atg ctc tcc acc ttc aga cca agc ggt gca actcca tac cat gat 672 Glu Met Leu Ser Thr Phe Arg Pro Ser Gly Ala Thr ProTyr His Asp 210 215 220 gcc tac gag aac gtc agg gag gtc atc caa ctt caagac cct ggt ctt 720 Ala Tyr Glu Asn Val Arg Glu Val Ile Gln Leu Gln AspPro Gly Leu 225 230 235 gag caa ctc aac gct tct cca gcc att gct ggt ttgatg cac cag gca 768 Glu Gln Leu Asn Ala Ser Pro Ala Ile Ala Gly Leu MetHis Gln Ala 240 245 250 255 tcc ttg ctc ggt atc aac gcc tcc atc tct cctgag ttg ttc aac atc 816 Ser Leu Leu Gly Ile Asn Ala Ser Ile Ser Pro GluLeu Phe Asn Ile 260 265 270 ttg act gag gag atc act gag ggc aac gct gaggag ttg tac aag aag 864 Leu Thr Glu Glu Ile Thr Glu Gly Asn Ala Glu GluLeu Tyr Lys Lys 275 280 285 aac ttc ggc aac att gag cca gcc tct ctt gcaatg cct gag tac ctc 912 Asn Phe Gly Asn Ile Glu Pro Ala Ser Leu Ala MetPro Glu Tyr Leu 290 295 300 aag agg tac tac aac ttg tct gat gag gag ctttct caa ttc att ggc 960 Lys Arg Tyr Tyr Asn Leu Ser Asp Glu Glu Leu SerGln Phe Ile Gly 305 310 315 aag gct tcc aac ttc ggt caa cag gag tac agcaac aac cag ctc atc 1008 Lys Ala Ser Asn Phe Gly Gln Gln Glu Tyr Ser AsnAsn Gln Leu Ile 320 325 330 335 act cca gtt gtg aac tcc tct gat ggc actgtg aag gtc tac cgc atc 1056 Thr Pro Val Val Asn Ser Ser Asp Gly Thr ValLys Val Tyr Arg Ile 340 345 350 aca cgt gag tac acc aca aac gcc tac caaatg gat gtt gag ttg ttc 1104 Thr Arg Glu Tyr Thr Thr Asn Ala Tyr Gln MetAsp Val Glu Leu Phe 355 360 365 cca ttc ggt ggt gag aac tac aga ctt gactac aag ttc aag aac ttc 1152 Pro Phe Gly Gly Glu Asn Tyr Arg Leu Asp TyrLys Phe Lys Asn Phe 370 375 380 tac aac gcc tcc tac ctc tcc atc aag ttgaac gac aag agg gag ctt 1200 Tyr Asn Ala Ser Tyr Leu Ser Ile Lys Leu AsnAsp Lys Arg Glu Leu 385 390 395 gtc agg act gag ggt gct cct caa gtg aacatt gag tac tct gcc aac 1248 Val Arg Thr Glu Gly Ala Pro Gln Val Asn IleGlu Tyr Ser Ala Asn 400 405 410 415 atc acc ctc aac aca gct gac atc tctcaa cca ttc gag att ggt ttg 1296 Ile Thr Leu Asn Thr Ala Asp Ile Ser GlnPro Phe Glu Ile Gly Leu 420 425 430 acc aga gtc ctt ccc tct ggc tcc tgggcc tac gct gca gcc aag ttc 1344 Thr Arg Val Leu Pro Ser Gly Ser Trp AlaTyr Ala Ala Ala Lys Phe 435 440 445 act gtt gag gag tac aac cag tac tctttc ctc ttg aag ctc aac aag 1392 Thr Val Glu Glu Tyr Asn Gln Tyr Ser PheLeu Leu Lys Leu Asn Lys 450 455 460 gca att cgt ctc agc aga gcc act gagttg tct ccc acc atc ttg gag 1440 Ala Ile Arg Leu Ser Arg Ala Thr Glu LeuSer Pro Thr Ile Leu Glu 465 470 475 ggc att gtg agg tct gtc aac ctt caactt gac atc aac act gat gtg 1488 Gly Ile Val Arg Ser Val Asn Leu Gln LeuAsp Ile Asn Thr Asp Val 480 485 490 495 ctt ggc aag gtc ttc ctc acc aagtac tac atg caa cgc tac gcc atc 1536 Leu Gly Lys Val Phe Leu Thr Lys TyrTyr Met Gln Arg Tyr Ala Ile 500 505 510 cat gct gag act gca ctc atc ctctgc aac gca ccc atc tct caa cgc 1584 His Ala Glu Thr Ala Leu Ile Leu CysAsn Ala Pro Ile Ser Gln Arg 515 520 525 tcc tac gac aac cag cct tcc cagttc gac agg ctc ttc aac act cct 1632 Ser Tyr Asp Asn Gln Pro Ser Gln PheAsp Arg Leu Phe Asn Thr Pro 530 535 540 ctc ttg aac ggc cag tac ttc tccact ggt gat gag gag att gac ctc 1680 Leu Leu Asn Gly Gln Tyr Phe Ser ThrGly Asp Glu Glu Ile Asp Leu 545 550 555 aac tct ggc tcc aca ggt gac tggaga aag acc atc ttg aag agg gcc 1728 Asn Ser Gly Ser Thr Gly Asp Trp ArgLys Thr Ile Leu Lys Arg Ala 560 565 570 575 ttc aac att gat gat gtc tctctc ttc cgt ctc ttg aag atc aca gat 1776 Phe Asn Ile Asp Asp Val Ser LeuPhe Arg Leu Leu Lys Ile Thr Asp 580 585 590 cac gac aac aag gat ggc aagatc aag aac aac ttg aag aac ctt tcc 1824 His Asp Asn Lys Asp Gly Lys IleLys Asn Asn Leu Lys Asn Leu Ser 595 600 605 aac ctc tac att ggc aag ttgctt gca gac atc cac caa ctc acc att 1872 Asn Leu Tyr Ile Gly Lys Leu LeuAla Asp Ile His Gln Leu Thr Ile 610 615 620 gat gag ttg gac ctc ttg ctcatt gca gtc ggt gag ggc aag acc aac 1920 Asp Glu Leu Asp Leu Leu Leu IleAla Val Gly Glu Gly Lys Thr Asn 625 630 635 ctc tct gca atc tct gac aagcag ttg gca acc ctc atc agg aag ttg 1968 Leu Ser Ala Ile Ser Asp Lys GlnLeu Ala Thr Leu Ile Arg Lys Leu 640 645 650 655 aac acc atc acc tcc tggctt cac acc cag aag tgg tct gtc ttc caa 2016 Asn Thr Ile Thr Ser Trp LeuHis Thr Gln Lys Trp Ser Val Phe Gln 660 665 670 ctc ttc atc atg acc agcacc tcc tac aac aag acc ctc act cct gag 2064 Leu Phe Ile Met Thr Ser ThrSer Tyr Asn Lys Thr Leu Thr Pro Glu 675 680 685 atc aag aac ctc ttg gacaca gtc tac cac ggt ctc caa ggc ttc gac 2112 Ile Lys Asn Leu Leu Asp ThrVal Tyr His Gly Leu Gln Gly Phe Asp 690 695 700 aag gac aag gct gac ttgctt cat gtc atg gct ccc tac att gca gcc 2160 Lys Asp Lys Ala Asp Leu LeuHis Val Met Ala Pro Tyr Ile Ala Ala 705 710 715 acc ctc caa ctc tcc tctgag aac gtg gct cac tct gtc ttg ctc tgg 2208 Thr Leu Gln Leu Ser Ser GluAsn Val Ala His Ser Val Leu Leu Trp 720 725 730 735 gct gac aag ctc caacct ggt gat ggt gcc atg act gct gag aag ttc 2256 Ala Asp Lys Leu Gln ProGly Asp Gly Ala Met Thr Ala Glu Lys Phe 740 745 750 tgg gac tgg ctc aacacc aag tac aca cca ggc tcc tct gag gct gtt 2304 Trp Asp Trp Leu Asn ThrLys Tyr Thr Pro Gly Ser Ser Glu Ala Val 755 760 765 gag act caa gag cacatt gtg caa tac tgc cag gct ctt gca cag ttg 2352 Glu Thr Gln Glu His IleVal Gln Tyr Cys Gln Ala Leu Ala Gln Leu 770 775 780 gag atg gtc tac cactcc act ggc atc aac gag aac gct ttc aga ctc 2400 Glu Met Val Tyr His SerThr Gly Ile Asn Glu Asn Ala Phe Arg Leu 785 790 795 ttc gtc acc aag cctgag atg ttc ggt gct gcc aca ggt gct gca cct 2448 Phe Val Thr Lys Pro GluMet Phe Gly Ala Ala Thr Gly Ala Ala Pro 800 805 810 815 gct cat gat gctctc tcc ctc atc atg ttg acc agg ttc gct gac tgg 2496 Ala His Asp Ala LeuSer Leu Ile Met Leu Thr Arg Phe Ala Asp Trp 820 825 830 gtc aac gct cttggt gag aag gct tcc tct gtc ttg gct gcc ttc gag 2544 Val Asn Ala Leu GlyGlu Lys Ala Ser Ser Val Leu Ala Ala Phe Glu 835 840 845 gcc aac tcc ctcact gct gag caa ctt gct gat gcc atg aac ctt gat 2592 Ala Asn Ser Leu ThrAla Glu Gln Leu Ala Asp Ala Met Asn Leu Asp 850 855 860 gcc aac ctc ttgctc caa gct tcc att caa gct cag aac cac caa cac 2640 Ala Asn Leu Leu LeuGln Ala Ser Ile Gln Ala Gln Asn His Gln His 865 870 875 ctc cca cct gtcact cca gag aac gct ttc tcc tgc tgg acc tcc atc 2688 Leu Pro Pro Val ThrPro Glu Asn Ala Phe Ser Cys Trp Thr Ser Ile 880 885 890 895 aac acc atcctc caa tgg gtc aac gtg gct cag caa ctc aac gtg gct 2736 Asn Thr Ile LeuGln Trp Val Asn Val Ala Gln Gln Leu Asn Val Ala 900 905 910 cca caa ggtgtc tct gct ttg gtc ggt ctt gac tac atc cag tcc atg 2784 Pro Gln Gly ValSer Ala Leu Val Gly Leu Asp Tyr Ile Gln Ser Met 915 920 925 aag gag acacca acc tac gct caa tgg gag aac gca gct ggt gtc ttg 2832 Lys Glu Thr ProThr Tyr Ala Gln Trp Glu Asn Ala Ala Gly Val Leu 930 935 940 act gct ggtctc aac tcc caa cag gcc aac acc ctc cat gct ttc ttg 2880 Thr Ala Gly LeuAsn Ser Gln Gln Ala Asn Thr Leu His Ala Phe Leu 945 950 955 gat gag tctcgc tct gct gcc ctc tcc acc tac tac atc agg caa gtc 2928 Asp Glu Ser ArgSer Ala Ala Leu Ser Thr Tyr Tyr Ile Arg Gln Val 960 965 970 975 gcc aaggca gct gct gcc atc aag tct cgc gat gac ctc tac caa tac 2976 Ala Lys AlaAla Ala Ala Ile Lys Ser Arg Asp Asp Leu Tyr Gln Tyr 980 985 990 ctc ctcatt gac aac cag gtc tct gct gcc atc aag acc acc agg atc 3024 Leu Leu IleAsp Asn Gln Val Ser Ala Ala Ile Lys Thr Thr Arg Ile 995 1000 1005 gctgag gcc atc gct tcc atc caa ctc tac gtc aac cgc gct ctt gag 3072 Ala GluAla Ile Ala Ser Ile Gln Leu Tyr Val Asn Arg Ala Leu Glu 1010 1015 1020aac gtt gag gag aac gcc aac tct ggt gtc atc tct cgc caa ttc ttc 3120 AsnVal Glu Glu Asn Ala Asn Ser Gly Val Ile Ser Arg Gln Phe Phe 1025 10301035 atc gac tgg gac aag tac aac aag agg tac tcc acc tgg gct ggt gtc3168 Ile Asp Trp Asp Lys Tyr Asn Lys Arg Tyr Ser Thr Trp Ala Gly Val1040 1045 1050 1055 tct caa ctt gtc tac tac cca gag aac tac att gac ccaacc atg agg 3216 Ser Gln Leu Val Tyr Tyr Pro Glu Asn Tyr Ile Asp Pro ThrMet Arg 1060 1065 1070 att ggt cag acc aag atg atg gat gct ctc ttg caatct gtc tcc caa 3264 Ile Gly Gln Thr Lys Met Met Asp Ala Leu Leu Gln SerVal Ser Gln 1075 1080 1085 agc caa ctc aac gct gac act gtg gag gat gccttc atg agc tac ctc 3312 Ser Gln Leu Asn Ala Asp Thr Val Glu Asp Ala PheMet Ser Tyr Leu 1090 1095 1100 acc tcc ttc gag caa gtt gcc aac ctc aaggtc atc tct gct tac cat 3360 Thr Ser Phe Glu Gln Val Ala Asn Leu Lys ValIle Ser Ala Tyr His 1105 1110 1115 gac aac atc aac aac gac caa ggt ctcacc tac ttc att ggt ctc tct 3408 Asp Asn Ile Asn Asn Asp Gln Gly Leu ThrTyr Phe Ile Gly Leu Ser 1120 1125 1130 1135 gag act gat gct ggt gag tactac tgg aga tcc gtg gac cac agc aag 3456 Glu Thr Asp Ala Gly Glu Tyr TyrTrp Arg Ser Val Asp His Ser Lys 1140 1145 1150 ttc aac gat ggc aag ttcgct gca aac gct tgg tct gag tgg cac aag 3504 Phe Asn Asp Gly Lys Phe AlaAla Asn Ala Trp Ser Glu Trp His Lys 1155 1160 1165 att gac tgc cct atcaac cca tac aag tcc acc atc aga cct gtc atc 3552 Ile Asp Cys Pro Ile AsnPro Tyr Lys Ser Thr Ile Arg Pro Val Ile 1170 1175 1180 tac aag agc cgcctc tac ttg ctc tgg ctt gag cag aag gag atc acc 3600 Tyr Lys Ser Arg LeuTyr Leu Leu Trp Leu Glu Gln Lys Glu Ile Thr 1185 1190 1195 aag caa actggc aac tcc aag gat ggt tac caa act gag act gac tac 3648 Lys Gln Thr GlyAsn Ser Lys Asp Gly Tyr Gln Thr Glu Thr Asp Tyr 1200 1205 1210 1215 cgctac gag ttg aag ttg gct cac atc cgc tac gat ggt acc tgg aac 3696 Arg TyrGlu Leu Lys Leu Ala His Ile Arg Tyr Asp Gly Thr Trp Asn 1220 1225 1230act cca atc acc ttc gat gtc aac aag aag atc agc gag ttg aag ttg 3744 ThrPro Ile Thr Phe Asp Val Asn Lys Lys Ile Ser Glu Leu Lys Leu 1235 12401245 gag aag aac cgt gct cct ggt ctc tac tgc gct ggt tac caa ggt gag3792 Glu Lys Asn Arg Ala Pro Gly Leu Tyr Cys Ala Gly Tyr Gln Gly Glu1250 1255 1260 gac acc ctc ttg gtc atg ttc tac aac cag caa gac acc cttgac tcc 3840 Asp Thr Leu Leu Val Met Phe Tyr Asn Gln Gln Asp Thr Leu AspSer 1265 1270 1275 tac aag aac gct tcc atg caa ggt ctc tac atc ttc gctgac atg gct 3888 Tyr Lys Asn Ala Ser Met Gln Gly Leu Tyr Ile Phe Ala AspMet Ala 1280 1285 1290 1295 tcc aag gac atg act cca gag caa agc aac gtctac cgt gac aac tcc 3936 Ser Lys Asp Met Thr Pro Glu Gln Ser Asn Val TyrArg Asp Asn Ser 1300 1305 1310 tac caa cag ttc gac acc aac aac gtc aggcgt gtc aac aac aga tac 3984 Tyr Gln Gln Phe Asp Thr Asn Asn Val Arg ArgVal Asn Asn Arg Tyr 1315 1320 1325 gct gag gac tac gag atc cca agc tctgtc agc tct cgc aag gac tac 4032 Ala Glu Asp Tyr Glu Ile Pro Ser Ser ValSer Ser Arg Lys Asp Tyr 1330 1335 1340 ggc tgg ggt gac tac tac ctc agcatg gtg tac aac ggt gac atc cca 4080 Gly Trp Gly Asp Tyr Tyr Leu Ser MetVal Tyr Asn Gly Asp Ile Pro 1345 1350 1355 acc atc aac tac aag gct gcctct tcc gac ctc aaa atc tac atc agc 4128 Thr Ile Asn Tyr Lys Ala Ala SerSer Asp Leu Lys Ile Tyr Ile Ser 1360 1365 1370 1375 cca aag ctc agg atcatc cac aac ggc tac gag ggt cag aag agg aac 4176 Pro Lys Leu Arg Ile IleHis Asn Gly Tyr Glu Gly Gln Lys Arg Asn 1380 1385 1390 cag tgc aac ttgatg aac aag tac ggc aag ttg ggt gac aag ttc att 4224 Gln Cys Asn Leu MetAsn Lys Tyr Gly Lys Leu Gly Asp Lys Phe Ile 1395 1400 1405 gtc tac acctct ctt ggt gtc aac cca aac aac agc tcc aac aag ctc 4272 Val Tyr Thr SerLeu Gly Val Asn Pro Asn Asn Ser Ser Asn Lys Leu 1410 1415 1420 atg ttctac cca gtc tac caa tac tct ggc aac acc tct ggt ctc aac 4320 Met Phe TyrPro Val Tyr Gln Tyr Ser Gly Asn Thr Ser Gly Leu Asn 1425 1430 1435 cagggt aga ctc ttg ttc cac agg gac acc acc tac cca agc aag gtg 4368 Gln GlyArg Leu Leu Phe His Arg Asp Thr Thr Tyr Pro Ser Lys Val 1440 1445 14501455 gag gct tgg att cct ggt gcc aag agg tcc ctc acc aac cag aac gct4416 Glu Ala Trp Ile Pro Gly Ala Lys Arg Ser Leu Thr Asn Gln Asn Ala1460 1465 1470 gcc att ggt gat gac tac gcc aca gac tcc ctc aac aag cctgat gac 4464 Ala Ile Gly Asp Asp Tyr Ala Thr Asp Ser Leu Asn Lys Pro AspAsp 1475 1480 1485 ctc aag cag tac atc ttc atg act gac tcc aag ggc acagcc act gat 4512 Leu Lys Gln Tyr Ile Phe Met Thr Asp Ser Lys Gly Thr AlaThr Asp 1490 1495 1500 gtc tct ggt cca gtg gag atc aac act gca atc agccca gcc aag gtc 4560 Val Ser Gly Pro Val Glu Ile Asn Thr Ala Ile Ser ProAla Lys Val 1505 1510 1515 caa atc att gtc aag gct ggt ggc aag gag caaacc ttc aca gct gac 4608 Gln Ile Ile Val Lys Ala Gly Gly Lys Glu Gln ThrPhe Thr Ala Asp 1520 1525 1530 1535 aag gat gtc tcc atc cag cca agc ccatcc ttc gat gag atg aac tac 4656 Lys Asp Val Ser Ile Gln Pro Ser Pro SerPhe Asp Glu Met Asn Tyr 1540 1545 1550 caa ttc aac gct ctt gag att gatggt tct ggc ctc aac ttc atc aac 4704 Gln Phe Asn Ala Leu Glu Ile Asp GlySer Gly Leu Asn Phe Ile Asn 1555 1560 1565 aac tct gct tcc att gat gtcacc ttc act gcc ttc gct gag gat ggc 4752 Asn Ser Ala Ser Ile Asp Val ThrPhe Thr Ala Phe Ala Glu Asp Gly 1570 1575 1580 cgc aag ttg ggt tac gagagc ttc tcc atc cca gtc acc ctt aag gtt 4800 Arg Lys Leu Gly Tyr Glu SerPhe Ser Ile Pro Val Thr Leu Lys Val 1585 1590 1595 tcc act gac aac gcactc acc ctt cat cac aac gag aac ggt gct cag 4848 Ser Thr Asp Asn Ala LeuThr Leu His His Asn Glu Asn Gly Ala Gln 1600 1605 1610 1615 tac atg caatgg caa agc tac cgc acc agg ttg aac acc ctc ttc gca 4896 Tyr Met Gln TrpGln Ser Tyr Arg Thr Arg Leu Asn Thr Leu Phe Ala 1620 1625 1630 agg caactt gtg gcc cgt gcc acc aca ggc att gac acc atc ctc agc 4944 Arg Gln LeuVal Ala Arg Ala Thr Thr Gly Ile Asp Thr Ile Leu Ser 1635 1640 1645 atggag acc cag aac atc caa gag cca cag ttg ggc aag ggt ttc tac 4992 Met GluThr Gln Asn Ile Gln Glu Pro Gln Leu Gly Lys Gly Phe Tyr 1650 1655 1660gcc acc ttc gtc atc cca cct tac aac ctc agc act cat ggt gat gag 5040 AlaThr Phe Val Ile Pro Pro Tyr Asn Leu Ser Thr His Gly Asp Glu 1665 16701675 agg tgg ttc aag ctc tac atc aag cac gtg gtt gac aac aac tcc cac5088 Arg Trp Phe Lys Leu Tyr Ile Lys His Val Val Asp Asn Asn Ser His1680 1685 1690 1695 atc atc tac tct ggt caa ctc act gac acc aac atc aacatc acc ctc 5136 Ile Ile Tyr Ser Gly Gln Leu Thr Asp Thr Asn Ile Asn IleThr Leu 1700 1705 1710 ttc atc cca ctt gac gat gtc cca ctc aac cag gactac cat gcc aag 5184 Phe Ile Pro Leu Asp Asp Val Pro Leu Asn Gln Asp TyrHis Ala Lys 1715 1720 1725 gtc tac atg acc ttc aag aag tct cca tct gatggc acc tgg tgg ggt 5232 Val Tyr Met Thr Phe Lys Lys Ser Pro Ser Asp GlyThr Trp Trp Gly 1730 1735 1740 cca cac ttc gtc cgt gat gac aag ggc atcgtc acc atc aac cca aag 5280 Pro His Phe Val Arg Asp Asp Lys Gly Ile ValThr Ile Asn Pro Lys 1745 1750 1755 tcc atc ctc acc cac ttc gag tct gtcaac gtt ctc aac aac atc tcc 5328 Ser Ile Leu Thr His Phe Glu Ser Val AsnVal Leu Asn Asn Ile Ser 1760 1765 1770 1775 tct gag cca atg gac ttc tctggt gcc aac tcc ctc tac ttc tgg gag 5376 Ser Glu Pro Met Asp Phe Ser GlyAla Asn Ser Leu Tyr Phe Trp Glu 1780 1785 1790 ttg ttc tac tac aca ccaatg ctt gtg gct caa agg ttg ctc cat gag 5424 Leu Phe Tyr Tyr Thr Pro MetLeu Val Ala Gln Arg Leu Leu His Glu 1795 1800 1805 cag aac ttc gat gaggcc aac agg tgg ctc aag tac gtc tgg agc cca 5472 Gln Asn Phe Asp Glu AlaAsn Arg Trp Leu Lys Tyr Val Trp Ser Pro 1810 1815 1820 tct ggt tac attgtg cat ggt caa atc cag aac tac caa tgg aac gtc 5520 Ser Gly Tyr Ile ValHis Gly Gln Ile Gln Asn Tyr Gln Trp Asn Val 1825 1830 1835 agg cca ttgctt gag gac acc tcc tgg aac tct gac cca ctt gac tct 5568 Arg Pro Leu LeuGlu Asp Thr Ser Trp Asn Ser Asp Pro Leu Asp Ser 1840 1845 1850 1855 gtggac cct gat gct gtg gct caa cat gac cca atg cac tac aag gtc 5616 Val AspPro Asp Ala Val Ala Gln His Asp Pro Met His Tyr Lys Val 1860 1865 1870tcc acc ttc atg agg acc ttg gac ctc ttg att gcc aga ggt gac cat 5664 SerThr Phe Met Arg Thr Leu Asp Leu Leu Ile Ala Arg Gly Asp His 1875 18801885 gct tac cgc caa ttg gag agg gac acc ctc aac gag gca aag atg tgg5712 Ala Tyr Arg Gln Leu Glu Arg Asp Thr Leu Asn Glu Ala Lys Met Trp1890 1895 1900 tac atg caa gct ctc cac ctc ttg ggt gac aag cca tac ctccca ctc 5760 Tyr Met Gln Ala Leu His Leu Leu Gly Asp Lys Pro Tyr Leu ProLeu 1905 1910 1915 agc acc act tgg tcc gac cca agg ttg gac cgt gct gctgac atc acc 5808 Ser Thr Thr Trp Ser Asp Pro Arg Leu Asp Arg Ala Ala AspIle Thr 1920 1925 1930 1935 act cag aac gct cat gac tct gcc att gtt gctctc agg cag aac atc 5856 Thr Gln Asn Ala His Asp Ser Ala Ile Val Ala LeuArg Gln Asn Ile 1940 1945 1950 cca act cct gct cca ctc tcc ctc aga tctgct aac acc ctc act gac 5904 Pro Thr Pro Ala Pro Leu Ser Leu Arg Ser AlaAsn Thr Leu Thr Asp 1955 1960 1965 ttg ttc ctc cca cag atc aac gag gtcatg atg aac tac tgg caa acc 5952 Leu Phe Leu Pro Gln Ile Asn Glu Val MetMet Asn Tyr Trp Gln Thr 1970 1975 1980 ttg gct caa agg gtc tac aac ctcaga cac aac ctc tcc att gat ggt 6000 Leu Ala Gln Arg Val Tyr Asn Leu ArgHis Asn Leu Ser Ile Asp Gly 1985 1990 1995 caa cca ctc tac ctc cca atctac gcc aca cca gct gac cca aag gct 6048 Gln Pro Leu Tyr Leu Pro Ile TyrAla Thr Pro Ala Asp Pro Lys Ala 2000 2005 2010 2015 ctt ctc tct gct gctgtg gct acc agc caa ggt ggt ggc aag ctc cca 6096 Leu Leu Ser Ala Ala ValAla Thr Ser Gln Gly Gly Gly Lys Leu Pro 2020 2025 2030 gag tcc ttc atgtcc ctc tgg agg ttc cca cac atg ttg gag aac gcc 6144 Glu Ser Phe Met SerLeu Trp Arg Phe Pro His Met Leu Glu Asn Ala 2035 2040 2045 cgt ggc atggtc tcc caa ctc acc cag ttc ggt tcc acc ctc cag aac 6192 Arg Gly Met ValSer Gln Leu Thr Gln Phe Gly Ser Thr Leu Gln Asn 2050 2055 2060 atc attgag agg caa gat gct gag gct ctc aac gct ttg ctc cag aac 6240 Ile Ile GluArg Gln Asp Ala Glu Ala Leu Asn Ala Leu Leu Gln Asn 2065 2070 2075 caggca gct gag ttg atc ctc acc aac ttg tcc atc caa gac aag acc 6288 Gln AlaAla Glu Leu Ile Leu Thr Asn Leu Ser Ile Gln Asp Lys Thr 2080 2085 20902095 att gag gag ctt gat gct gag aag aca gtc ctt gag aag agc aag gct6336 Ile Glu Glu Leu Asp Ala Glu Lys Thr Val Leu Glu Lys Ser Lys Ala2100 2105 2110 ggt gcc caa tct cgc ttc gac tcc tac ggc aag ctc tac gatgag aac 6384 Gly Ala Gln Ser Arg Phe Asp Ser Tyr Gly Lys Leu Tyr Asp GluAsn 2115 2120 2125 atc aac gct ggt gag aac cag gcc atg acc ctc agg gcttcc gca gct 6432 Ile Asn Ala Gly Glu Asn Gln Ala Met Thr Leu Arg Ala SerAla Ala 2130 2135 2140 ggt ctc acc act gct gtc caa gcc tct cgc ttg gctggt gca gct gct 6480 Gly Leu Thr Thr Ala Val Gln Ala Ser Arg Leu Ala GlyAla Ala Ala 2145 2150 2155 gac ctc gtt cca aac atc ttc ggt ttc gct ggtggt ggc tcc aga tgg 6528 Asp Leu Val Pro Asn Ile Phe Gly Phe Ala Gly GlyGly Ser Arg Trp 2160 2165 2170 2175 ggt gcc att gct gag gct acc ggt tacgtc atg gag ttc tct gcc aac 6576 Gly Ala Ile Ala Glu Ala Thr Gly Tyr ValMet Glu Phe Ser Ala Asn 2180 2185 2190 gtc atg aac act gag gct gac aagatc agc caa tct gag acc tac aga 6624 Val Met Asn Thr Glu Ala Asp Lys IleSer Gln Ser Glu Thr Tyr Arg 2195 2200 2205 agg cgc cgt caa gag tgg gagatc caa agg aac aac gct gag gca gag 6672 Arg Arg Arg Gln Glu Trp Glu IleGln Arg Asn Asn Ala Glu Ala Glu 2210 2215 2220 ttg aag caa atc gat gctcaa ctc aag tcc ttg gct gtc aga agg gag 6720 Leu Lys Gln Ile Asp Ala GlnLeu Lys Ser Leu Ala Val Arg Arg Glu 2225 2230 2235 gct gct gtc ctc cagaag acc tcc ctc aag acc caa cag gag caa acc 6768 Ala Ala Val Leu Gln LysThr Ser Leu Lys Thr Gln Gln Glu Gln Thr 2240 2245 2250 2255 cag tcc cagttg gct ttc ctc caa agg aag ttc tcc aac cag gct ctc 6816 Gln Ser Gln LeuAla Phe Leu Gln Arg Lys Phe Ser Asn Gln Ala Leu 2260 2265 2270 tac aactgg ctc aga ggc cgc ttg gct gcc atc tac ttc caa ttc tac 6864 Tyr Asn TrpLeu Arg Gly Arg Leu Ala Ala Ile Tyr Phe Gln Phe Tyr 2275 2280 2285 gacctt gct gtg gcc agg tgc ctc atg gct gag caa gcc tac cgc tgg 6912 Asp LeuAla Val Ala Arg Cys Leu Met Ala Glu Gln Ala Tyr Arg Trp 2290 2295 2300gag ttg aac gat gac tcc gcc agg ttc atc aag cca ggt gct tgg caa 6960 GluLeu Asn Asp Asp Ser Ala Arg Phe Ile Lys Pro Gly Ala Trp Gln 2305 23102315 ggc acc tac gct ggt ctc ctt gct ggt gag acc ctc atg ctc tcc ttg7008 Gly Thr Tyr Ala Gly Leu Leu Ala Gly Glu Thr Leu Met Leu Ser Leu2320 2325 2330 2335 gct caa atg gag gat gct cac ctc aag agg gac aag agggct ttg gag 7056 Ala Gln Met Glu Asp Ala His Leu Lys Arg Asp Lys Arg AlaLeu Glu 2340 2345 2350 gtg gag agg aca gtc tcc ctt gct gag gtc tac gctggt ctc cca aag 7104 Val Glu Arg Thr Val Ser Leu Ala Glu Val Tyr Ala GlyLeu Pro Lys 2355 2360 2365 gac aac ggt cca ttc tcc ctt gct caa gag attgac aag ttg gtc agc 7152 Asp Asn Gly Pro Phe Ser Leu Ala Gln Glu Ile AspLys Leu Val Ser 2370 2375 2380 caa ggt tct ggt tct gct ggt tct ggt aacaac aac ttg gct ttc ggc 7200 Gln Gly Ser Gly Ser Ala Gly Ser Gly Asn AsnAsn Leu Ala Phe Gly 2385 2390 2395 gct ggt act gac acc aag acc tcc ctccaa gcc tct gtc tcc ttc gct 7248 Ala Gly Thr Asp Thr Lys Thr Ser Leu GlnAla Ser Val Ser Phe Ala 2400 2405 2410 2415 gac ctc aag atc agg gag gactac cca gct tcc ctt ggc aag atc agg 7296 Asp Leu Lys Ile Arg Glu Asp TyrPro Ala Ser Leu Gly Lys Ile Arg 2420 2425 2430 cgc atc aag caa atc tctgtc acc ctc cca gct ctc ttg ggt cca tac 7344 Arg Ile Lys Gln Ile Ser ValThr Leu Pro Ala Leu Leu Gly Pro Tyr 2435 2440 2445 caa gat gtc caa gcaatc ctc tcc tac ggt gac aag gct ggt ttg gcg 7392 Gln Asp Val Gln Ala IleLeu Ser Tyr Gly Asp Lys Ala Gly Leu Ala 2450 2455 2460 aac ggt tgc gaggct ctt gct gtc tct cat ggc atg aac gac tct ggt 7440 Asn Gly Cys Glu AlaLeu Ala Val Ser His Gly Met Asn Asp Ser Gly 2465 2470 2475 caa ttc caactt gac ttc aac gat ggc aag ttc ctc cca ttc gag ggc 7488 Gln Phe Gln LeuAsp Phe Asn Asp Gly Lys Phe Leu Pro Phe Glu Gly 2480 2485 2490 2495 attgcc att gac caa ggc acc ctc acc ctc tcc ttc cca aac gct tcc 7536 Ile AlaIle Asp Gln Gly Thr Leu Thr Leu Ser Phe Pro Asn Ala Ser 2500 2505 2510atg cca gag aag gga aag caa gcc acc atg ctc aag acc ctc aac gat 7584 MetPro Glu Lys Gly Lys Gln Ala Thr Met Leu Lys Thr Leu Asn Asp 2515 25202525 atc atc ctc cac atc agg tac acc atc aag tgagctc 7621 Ile Ile LeuHis Ile Arg Tyr Thr Ile Lys 2530 2535

We claim:
 1. An isolated nucleic acid of SEQ ID NO: 3 or SEQ ID NO:4. 2.A transgenic monocot cell having a genome comprising SEQ ID NO:3 or SEQID NO:4.
 3. A transgenic dicot cell having a genome comprising SEQ IDNO:3 or SEQ ID NO:4.
 4. A transgenic plant with a genome comprising anucleic acid of SEQ ID NO: 3 or SEQ ID NO:4 that imparts insectresistance.
 5. A transgenic plant of claim 4 wherein the plant is rice.6. A transgenic plant of claim 4 wherein the plant is maize.
 7. Atransgenic plant of claim 4 wherein the plant is tobacco.